RU2725884C2 - Deoxyuridine triphosphates marked with zwitterionic indocyanine dyes - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: present invention refers to used as fluorescent labels for enzymatic marking DNA during PCR nucleoside triphosphate and intermediate compounds for their production. Disclosed are compounds of formulas:

,

,

where Y is -(CH₂)₃-; -(CH₂)₅; -(CH₂)₅CONH(CH₂)₃-; X is -CH₂-; -CH₂CONH(CH₂)₅-; -CH₂CONH(CH₂)₅CONH(CH₂)₅-; M⁺ represents H⁺, Na⁺, Li⁺, K⁺, [CH₃(CH₂)_n]_(4-m)NH_m ⁺, where n=0–5, m=0–4. Disclosed are compounds of formulas:

,

,

,

where R is -OH; -NH(CH₂)₅COOH; -NH(CH₂)₅CONH(CH₂)₅COOH; m=3–5. Disclosed is a compound of formula

,

where R is -NH(CH₂)₅COOH; -NH(CH₂)₅CONH(CH₂)₅COOH at m=3 or R is -OH; -NH(CH₂)₅COOH; -NH(CH₂)₅CONH(CH₂)₅COOH at m=5.

EFFECT: disclosed are novel compounds which are effective as fluorescent labels for enzymatic marking of DNA during PCR, as well as novel compounds for preparing said compounds.

13 cl, 43 ex, 3 tbl, 5 dwg

Description

FIELD OF THE INVENTION

The present invention relates to the field of organic and bioorganic chemistry, molecular biology, biotechnology and medicine, namely, to new modified nucleoside triphosphates containing a zwitterionic cyanine dye attached via a trans-alkene linker of various structures. The proposed compounds and materials based on them can be used as fluorescent labels for enzymatic labeling of DNA during PCR.

State of the art

Modern methods for detecting and identifying the causes and causative agents of diseases are increasingly based on molecular genetic analysis. The labeling procedure for visualizing molecular genetic analysis results is an important and integral step in the process (Ranasinghe R.T., Brown T. Fluorescent based strategies for genetic analysis. Chem. Commun. 2005. P. 5487-5502). Increasing the sensitivity and accuracy of molecular genetic analysis is one of the main directions in the development of medical diagnostics. Sensitivity is limited by intrinsic, background, fluorescence of biological material.

Nucleoside triphosphates marked with cyanine dyes of the near IR range, as well as the transition to the region with lower background fluorescence, to the infrared region, to the wavelength range of 700-900 nm, are of particular interest due to the extremely high sensitivity of their registration, which allows them to be used as an instrument to solve the problems of molecular biology and medicine. Dyes differ in the nature and arrangement of substituents and functional groups, which affects the efficiency of fluorescently-labeled nucleoside triphosphates in incorporating into DNA synthesized during PCR under the influence of matrix-dependent DNA polymerases.

Despite the variety of cyanine dyes of various structures presented in the literature, the number of proposed fluorescently-labeled nucleoside triphosphates is several times smaller. The authors of the patent (Fluorescent nucleotides containing a cyanine, merocyanine or styryl dye for the detection of nucleic acid, European patent, 1152008, - 2001, Fuji photo film co., Japan) proposed using cyanine dyes with a sulfonamide substituent at position 5 of indoleninium to mark nucleoside triphosphates. fragment.

The patent (Improved process and method for the preparation of asymmetric monofunctionalized indocyanine labelling reagents and obtained compounds, European patent, 1211294, - 2002, Innosense S.r.l, Italy) proposed the synthesis of fluorescently-labeled dUTP by adding water-soluble cyanine dyes with different amounts of sulfo groups.

In the patent (Modified carbocyanine dyes and their conjugates, European patent, 1801165, - 2007, Molecular Probes Inc., USA), Molecular Probes proposed indodicarbocyanine dyes with a carboxypentyl group at position 3 of the indolenin fragment and their conjugates with nucleoside triphosphates.

The patent (Cyanine dye labeling reagents with meso-substitution, US patent, 2004186278, - 2004, Amersham Biosciences Corp., USA) presents the synthesis of fluorescently-labeled dUTP using cyanine dyes with a substituent in the meso position of the polymethine chain.

The patent (New fluorescence cyanine labels containing a sulfamido linker arm, European patent, 1491591, - 2004, Visen medical inc., Italy) presents a series of nucleoside triphosphates labeled with cyanine dyes with a carboxyl group attached via a sulfamidoalkyl chain in position 5.

The IR-Indotricarbocyanine dyes are already widely used for medical research and their field of application is constantly expanding. On the one hand, there is a search for conjugates of known dyes with new receptor groups, and on the other hand, a search for new dyes with increased photostability and increased fluorescence efficiency. Currently, firms sell infrared fluorescent dyes abroad. It should be noted that the number of dyes offered is several times smaller than the commercially available dyes in the visible range. In addition, the structure and spectral characteristics of the proposed dyes are most often not represented or limited by the wavelengths of excitation and fluorescence.

The dye Su7®, offered by General Electric Healthcare Life Sciences, contains two sulfo groups and a heptamethylene chain between indolenine fragments. This dye has a fairly low photostability and chemoresistance.

The authors of the patent (Heat development image forming process and heptamethine cyanine dye compound, US patent, 6072059, - 2000, Fuji Photo Film Co., Japan) synthesized heptamethine dyes containing a cycloheptene fragment in the meso position of the chain.

Li-Cor patented indotricarbocyanine dyes with a cyclohexene fragment in the meso position of the heptamethylene chain (Cyanine dye compounds and labaling methods, US patent, 6593148, - 2003, Li-Cor Inc., USA; Optical fluorescent imaging using cyanine dye, WO 2007 / 2007136996, Li-Cor Inc., USA). These dyes, due to their spectral-luminescent properties, are widely used for biomedical research.

The patent (Fluorescent cyanine labels containing sulfamido linker arm, US patent, 6448008, - 2002, Innosense Srl, Italy) presents a synthesis of a number of indotricarbocyanine dyes containing a cyclohexene fragment in the meso position and a reactive group at position 5 attached via a sulfamidoalkyl chain .

The patent (Heptamethine cyanine compound, near infrared absorbing ink, near infrared absorbing sheet and silver halide photographic material, US patent, 5973158, - 1999, Fuji Photo Film Co. Ltd., Japan) describes the synthesis of heptamethine dyes containing in the meso position 4-pyridyl chain.

GE Heakthcare UK Limited proposed the synthesis of indocyanine dyes containing a sulfonate-butyl group at position 3 of the indolenin moiety (Water-soluble fluoro-substituted cyanine dyes as reactive fluorescence labeling reagents, US patent, 7767829, - 2010, GE Heakthcare UK Limited, Great Britain).

Any new approaches that allow controlling the luminescent properties of cyanines are of undoubted interest, both for science and for practice. The limitations of existing synthetic methods, as well as data on the influence of the location and nature of the substituents on the spectral properties, are clearly visible in the series of zwitterionic cyanine dyes containing trimethylammonium alkyl substituent in their structure. The preparation of new indocyanine dyes, as well as their conjugates with nucleoside triphosphates, can make it possible to find new regular relationships between their structure, properties, and ability to efficiently integrate into DNA synthesized during PCR under the influence of matrix-dependent DNA polymerases.

Disclosure of invention

The introduction of fluorescently-labeled nucleoside triphosphates using DNA polymerases during PCR has been widely used for enzymatic labeling of DNA. The effectiveness of the labeling process greatly depends on the type of dye introduced, on the structure and length of the linker linking the nucleic base and the fluorophore. As labeled substrates for DNA polymerases based on 2'-deoxyuridine-5'-triphosphate, its derivatives with linkers based on E-ethene (trans-ethylene isomer) are a good option.

The aim of the present invention was the synthesis of new fluorescently-labeled nucleoside triphosphates containing at position 5 a zwitterionic disulfonated indocarbocyanine dye with a trimethylammoniumalkyl substituent, the fluorophore being attached via linkers of various structures, including a trans-alkene group, and their use for enzymatic DNA labeling.

This goal is achieved by the structure of the proposed new type of fluorescently-labeled nucleoside triphosphates of General formula (A).

Where:

X represents —CH ₂ -; -CH ₂ CONH (CH ₂ ) ₅ -; -CH ₂ CONH (CH ₂ ) ₅ CONH (CH ₂ ) ₅ -;

Y represents - (CH ₂ ) ₃ -; - (CH ₂ ) ₅ -; - (CH ₂ ) ₅ CONH (CH ₂ ) ₃ -;

n = 1-3

M ⁺ represents H ⁺ , Na ⁺ , Li ⁺ , K ⁺ , [CH ₃ (CH ₂ ) _n ] ₍₄ - _m ) NH _m ⁺ (where n = 0-5, m = 0-4).

The proposed fluorescently-labeled triphosphates of the general formula (A) are characterized by the fact that the structure of the zwitterionic indocyanine dye of the general formula (B), attached via a transalkene linker of various lengths, contains two negatively charged sulfo groups and a positively charged trimethylammonium alkyl substituent.

Where:

R represents —OH; -NH (CH ₂ ) ₅ COOH; -NH (CH ₂ ) ₅ CONH (CH ₂ ) ₅ COOH;

Y represents - (CH ₂ ) ₃ -; - (CH ₂ ) ₅ -; - (CH ₂ ) ₅ CONH (CH ₂ ) ₃ -;

n = 1-3

In particular, compounds conventionally assigned to compounds of group C corresponding to the general formula (C)

Where:

R represents —OH; -NH (CH ₂ ) ₅ COOH; -NH (CH ₂ ) ₅ CONH (CH ₂ ) ₅ COOH;

n = 1-3

as well as compounds conventionally assigned to compounds of group D corresponding to the general formula (D)

Where:

R represents —OH; -NH (CH ₂ ) ₅ COOH; -NH (CH ₂ ) ₅ CONH (CH ₂ ) ₅ COOH;

m = 3-5

n = 1-3

The indicated structural differences between the zwitterionic indocyanine dyes of the general formulas (C) and (D), which are part of the nucleoside triphosphates (A), lead to a number of distinctive properties, namely, the electroneutrality of the fluorophore molecule due to the presence of two negatively charged sulfo groups and a positively charged trimethylammonium alkyl Deputy to increased water solubility, increased chemical resistance and heat resistance. Changing the structure of the chromophore and introducing a linker of different lengths has a positive effect on the efficiency of incorporation of modified nucleoside triphosphates into the growing DNA chain during PCR. To obtain a linker connecting the dye molecule and dUTP, aminocaproic acid was used.

The spectral-luminescent characteristics of the dyes of the general formula (B) (m = 2) in aqueous phosphate-buffered saline (PBS) (10 mM potassium phosphate buffer, 0.9% NaCl, pH 7.4) were determined. The values of the molar extinction coefficient of dyes in some cases reach ~ 250,000 (Table 1, Table 2). The fluorescence quantum yield of dyes of the general formula (B) (m = 2) in PBS exceeds 30% (Table 1, Table 2). The proposed dyes of structures (B) differ in the spectral range of excitation and fluorescence.

The spectral-luminescent characteristics of dyes of the general formula (B) (m = 3) in aqueous phosphate-buffered saline (PBS) (10 mM potassium phosphate buffer, 0.9% NaCl, pH 7.4) were determined. The values of the molar extinction coefficient of dyes in some cases reach ~ 250,000 (Table 1, Table 2). The quantum fluorescence yield of dyes of the general formula (B) (m = 3) in PBS exceeds 35% (Table 1, Table 2).

A Jasco V-550 spectrophotometer (Japan) recorded absorption spectra of dyes of the general formula (B) (m = 2, 3) in PBS solutions at 20 ° C (Fig. 1, Fig. 2). The absorption spectrum of the synthesized dyes is represented by an intense band and a low-intensity shoulder, which is located on the short-wave side of the band. The Cary Eclips spectrofluorimeter recorded the excitation and fluorescence spectra of solutions of synthesized dyes of the general formula (B) (m = 2, 3) in PBS at 20 ° C (Fig. 1, Fig. 2, Table 1, Table 2). The introduction of the ammonium group in position 1 had practically no effect on the maxima of excitation and fluorescence compared with the commercially available dye Su5 and Su7, respectively.

The thermal stability of the dyes of the general formula (B) was determined (m = 2, 3) (Fig. 3). The experimental results in the case of dyes of the general formula (B) (m = 3) were compared with the data obtained for dyes (Cy7) and (Cy7-monosulfo), since heptamethine dyes are less thermo- and light-resistant compared to pentamethine analogues.

The thermal stability of the dyes was investigated on the basis of data on the optical density of their aqueous solutions with a concentration of 10 ^-6 mol / L. Previously, the dye in the chromatographic purification process deposited on RP-18 sorbent was washed with a 0.01 M EDTA solution. This procedure allowed the removal of heavy metal ions that can catalyze the process of thermal oxidative degradation. Bidistilled water (MQ) was used as a solvent. The absorption spectra of dye solutions were recorded on a Jasco V-550 spectrophotometer (Japan). The data obtained were normalized and then used the concept of a normalized absorption value (D _norms ).

Dye solutions in MQ with a concentration of 10 ^-6 mol / L were heated in a thermostat (AccuBlock Digital Dry Bath, Labnet, Switzerland, 2 ml Eppendorf tube block) for 6 h at a temperature of 92 ° C. Registration of optical density was carried out at regular intervals. As the dyes were heated in MQ, the optical density decreased. Thus, the degree of destruction of the chromophore dye system was judged by a change in the optical density. According to the data obtained, a graph of the dependence of D _norms on time was constructed (Fig. 3). Based on the obtained experimental data, it is seen that the dye of the general formula (C) (m = 2) has high thermal stability. The dye of the general formula (C) (m = 3), compared with the electrically neutral monosulfonated dye (Cy7-monosulfo), has enhanced thermal stability comparable to the data obtained for the disulfurized dye (Cy7).

The light fastness (photo stability) of the dyes of the general formula (C) was determined (m = 2, 3). Photostability should be sufficient for the practical use of synthesized fluorophores in the study of biomolecules. In studying the light fastness, we used dyes purified from traces of heavy metals by treatment with a 0.01 M EDTA solution. The light fastness of the synthesized dyes was studied by irradiating their solutions in MQ with a concentration of 10 ^-6 mol / L in a box isolated from external light and a change in the optical density of the solution was recorded. A 60 W incandescent lamp located at the same level as the test samples at a distance of 30 cm was used as a light source. The prepared dye solutions were placed in glass vials and irradiated for 6 h, recording optical density at regular intervals. The data obtained were normalized to the maximum value obtained for each dye, and the dependence of D _norms on time was plotted (Fig. 4).

Our studies showed that the introduction of a trimethylammonium alkyl substituent at position 1 of the indotricarbocyanine dye of the general formula (C) (m = 3) led to an increase in photostability compared to the dye (Cy7-monosulfo). In terms of the combination of heat and light fastness, fluorescence brightness (the value of the molar extinction coefficient and fluorescence quantum yield), as well as chemical stability, dyes of the general formula (C) (m = 3) (using the example of dye C4) with an ammonium substituent in position 1 surpasses the dye ( Cy7-monosulfo).

The following are the results of the use of dyes to obtain fluorescently labeled nucleoside triphosphates of the general formula (A), followed by their use for enzymatic labeling of DNA during PCR.

All dyes of the general formula (B) in the form of their / para-nitrophenyl esters with a yield of 60-70% (depending on the structure of the dye used (B)) label nucleotides containing an aminoallyl substituent in the C5 position. The reaction can be carried out followed by the replacement of anions in the compounds of the invention of general formula (A), treatment with acids, acid salts with other counterions (or without replacing the anion). Varying the listed counterions does not affect the spectral-luminescent characteristics of the nucleotides of the general formula (A). Therefore, a detailed description of the properties of modified nucleotides of the general formulas (A, M ⁺ = Na ⁺ ) is presented using their sodium salts as an example.

Nucleotides of the general formula (A) modified with dyes with a trimethylammonium alkyl substituent are characterized by intense absorption and fluorescence bands and a fluorescence quantum yield of 30-34% in PBS (Table 3).

It was found that the results of the polymerase chain reaction using fluorescently-labeled nucleoside triphosphates of the general formula (A) depend on the structure of the modified nucleotides used as substrates in PCR. The efficiency of incorporation of modified nucleotides of general formula (A) under PCR conditions was analyzed using the KRAS-biochip test system (Biochip-IMB, Moscow, Russia), designed to determine mutations characteristic of pancreatic cancer (M.A. Emelyanova. Determination of mutations in the KRAS gene in tumor cells using biological microarrays // Molecular Biology. 2011. - V. 45, No. 5. - S. 863-870).

To assess the degree of incorporation of modified nucleotides by Taq polymerase into DNA, the average fluorescence signal of perfect duplexes in the biochip cells was calculated and then normalized to the value obtained for a commercially available product (AAdUTP-Cy5 - Jena Bioscience GmbH, Germany, NU-803-CY5- S). The fluorescent signal was found to be maximal for nucleotides A10-A18 containing zwitterionic indotricarbocyanine dye. For both groups of modified nucleoside triphosphates A1-A9 and A10-A18, the dependence of the normalized fluorescence signal on the length of the linker separating the fluorophore from the pyrimidine base, which varies in the series: (A3)> (A2)> (A1); (A6)> (A5)> (A4); (A9)> (A8)> (A7); (A12)> (A11)> (A10); (A15)> (A14)> (A13); (A18)> (A17)> (A16). The fluorescent signals obtained for nucleotides (A) with a long linker containing in their structure a dye of the general formulas (C) and (D, m = 3) turned out to be significantly higher than the signals obtained using commercially available AAdUTP-Cy5.

It was found that all dyes of formulas (B) in the conjugates with nucleotides do not inhibit the polymerase chain reaction. Modified nucleotides of the general formula (A) do not affect the stability of all other components of the analysis. It was found that all compounds of formula (A) are stable enough to conduct a complete analysis cycle without noticeable dye decomposition. The use of fluorescence-labeled nucleotides of the general formula (A) in PCR and registration of fluorescence in the infrared region of the spectrum allows to obtain a fluorescent signal 20-25 times higher compared to the commercially available product AAdUTP-Cy5 in determining mutations of pancreatic cancer using the test - KRAS-biochip systems (Biochip-IMB, Moscow, Russia).

By the combination of all the properties of the compounds of formula (A), they can be successfully used for enzymatic labeling of DNA during PCR, as well as fluorescent labels in the development of diagnostic test systems based on PCR.

Brief Description of the Figures

Table 1. Spectral characteristics of indocyanine dyes of the general formula (C) in PBS (+ 25 ° C). Quantum fluorescence yields were determined relative to commercially available Cy5 (Q = 28% (PBS), Amersham, GE Healthcare).

Table 2. Spectral characteristics of indocyanine dyes of the general formula (D) in PBS (+ 25 ° C). Quantum fluorescence yields were determined relative to commercially available Cy5 (Q = 28% (PBS), Amersham, GE Healthcare).

Table 3. Spectral characteristics of fluorescently-labeled nucleoside triphosphates of the general formula (A) in PBS (+ 25 ° C). Quantum fluorescence yields were determined relative to the commercially available Cy5-dUTP (Amersham, GE Healthcare PA55022). M ⁺ - H ⁺ , Na ⁺ , Li ⁺ , K ⁺ , [CH ₃ (CH ₂ ) _x ] _(4-y) NH _y ⁺ (where x = 0-5, y = 0-4).

FIG. 1. Normalized absorption and fluorescence spectra of indocyanine dyes of the general formula (B, n = 3).

FIG. 2. Normalized absorption and fluorescence spectra of indocyanine dyes of the general formula (B, n = 2).

FIG. 3. The graph of the dependence of D _norms on time when heating solutions of indocyanine dyes of the General formula (C) in MQ at 92 ° C.

FIG. 4. The graph of the dependence of D _norms on time when irradiating indocyanine dyes of the General formula (C)

FIG. 5. Normalized fluorescence signal for modified nucleoside triphosphates of the general formula (A) using the KRAS-biochip diagnostic test system (Biochip-IMB, Moscow, Russia).

The implementation of the invention

The compounds of the invention of general formula (A) were synthesized according to Scheme 1.

The synthesis of asymmetric indodicarbocyanine dyes of the general formula (D) that are part of nucleoside triphosphates (A) is based on the condensation reaction of indoleninium salts containing an active methylene group and dianyl (Scheme 2) (V.E. Kuznetsova et al., Izv. AN, Ser Chem., 2007, No. 11, pp. 2186-2190; V.E. Kuznetsova et al., Izv. AN, Ser.chem., 2007, No. 12, pp. 2355-2359; VE Kuznetsova et al, Mend Comm. 2008, No. 3, pp. 138-140). The reaction with NN-diphenylformamidine (3, n = 1), with malonic dialdehyde dianyl hydrochloride (3, n = 2), with glutacone dialdehyde dianyl hydrochloride (3, n = 3) leads to the formation of dyes (D, n = 1) , (D, n = 2) and (D, n = 3), respectively. In the course of the reaction, in addition to the target compound, two more symmetrical by-products are formed. In the first stage, indolenine containing a quaternary ammonium group at position 1 was introduced into the condensation reaction. The second stage of the preparation of indodicarbocyanine dye was carried out using anhydrous potassium acetate as a condensing agent. For a complete reaction to occur, heating at 118 ° C for 1-3 hours is sufficient.

The synthesis of intermediates necessary for the preparation of dyes of formula (B) is shown in Scheme 4 (Maksim A. Spitsyn, Viktoriya E. Kuznetsova, Valeriy E. Shershov, Marina A. Emelyanova, Teimur O. Guseinov, Sergey A. Lapa, Tatyana V Nasedkina, Alexander S. Zasedatelev, Alexander V. Chudinov, Synthetic route to novel zwitterionic pentamethine indocyanine fluorophores with various substitutions // Dyes and Pigments, V. 147, P. 199-210). Condensation of phenylhydrazine (6) with methylisopropyl ketone gave the corresponding indolenine (7) (Scheme 3). N-Alkyl derivatives (compound (2)) were obtained by the action of (3-bromopropyl) trimethyl ammonium bromide or (5-bromopentyl) trimethyl ammonium bromide in butyronitrile, respectively.

Another way to obtain zwitterionic cyanine dyes with a trimethylammoniumalkyl substituent is to modify the symmetric dye (5) containing two carboxyl groups. For this, a symmetric dye (5) was synthesized (Scheme 3). Indocyanine dye (5, n = 1, 2) is obtained in one step by condensation of the indoleninium salt and dianyl (3, n = 1, 2). Whereas the synthesis of indotricarbocyanine dye (5, n = 3) is carried out in two stages using a three-fold excess (with respect to the derivative of glutaconic aldehyde (3, n = 3)) of quaternized indolenine (4), which was introduced at each stage in equal portions. The reaction was carried out in a mixture of acetic anhydride - acetic acid with the addition of potassium acetate as a condensing agent.

Several methods were used to obtain the activated dye ester (5). The first method was to activate the carboxyl group of the dye (5) using HBTU and N-hydroxyphthalimide in DMF with the addition of DIPEA, while we observed incomplete conversion of the starting acid (5) and the formation of two activated esters with the predominant formation of a mono product. It should be noted that at room temperature the reaction time was 3 days. The use of N, N'-carbonyldiimidazole instead of HBTU led to a sharp decrease in the rate of formation of activated esters up to 10 days. The reaction in which HBTU acted as a condensing agent and NHS as an activator was much more selective and faster, and the product of di-addition was mainly formed. The progress of the reaction was monitored by TLC on RP-18 plastics.

After the formation of a mixture of mono- and di-activated esters (without isolation), a solution of 3- (dimethylamino) -1-propylamine dihydrochloride in a carbonate-bicarbonate buffer solution was added to the reaction mixture (Scheme 3). For the complete reaction to take place, the reaction mixture must be stirred for 24 hours and 72 hours at room temperature (in the case of succinimide and phthalimide esters, respectively). When succinimide esters were used, the amine addition reaction proceeded more selectively with the predominant formation of the mono addition product (6). While the rate of hydrolysis of phthalimide esters was higher than the rate of formation of the amide bond and the product (6) was formed with a yield of <10%.

The quaternization of the product (6) was carried out using methyl iodide (for dyes (C, n = 1, 2) and methyl ester of p-toluenesulfonic acid (for dye (C, n = 3) in DMF at room temperature for 3 h. During The reaction using p-toluenesulfonic acid methyl ester resulted in the predominant formation of a quaternized product (C, R = OH, n = 3)) and trace amounts of dye methyl ester. In the case of the use of methyl iodide as an alkylating agent, the predominant formation of methyl ester (C, R = OCH ₃ ,), which was converted into the corresponding acid by acid hydrolysis. In the case of dye (C4), acid hydrolysis is impossible due to the destruction of the chromophore part of the fluorophore.

To change the length of the linker separating the fluorophore from the nucleoside triphosphate, conjugates of dyes with aminocaproic acid were obtained. For this, activated dye esters of the general formula (B) were preliminarily obtained using p-nitrophenol and HBTU in DMF (for dyes (B, n = 1, 2) or by the action of bis-p-nitrophenyl carbonate in DMSO (for dyes (B, n = 3) Next, the obtained activated ester was treated with a solution of aminocaproic acid in carbonate-bicarbonate buffer solution.

All synthesized dyes were isolated by reverse phase chromatography on a C18-RP column in an acetonitrile-0.1 M TEAA system in a linear concentration gradient from 10 to 50% acetonitrile. The structure and identity of compound (16) was confirmed by TLC, mass spectrometry (MALDI-TOF), and ¹ H NMR spectroscopy.

For the synthesis of fluorescently-labeled AAdUTP, we previously obtained active derivatives of dyes of the general formula (B). Active derivatives were prepared by esterification of the dyes of general formula (B) with p-nitrophenol in the presence of HBTU in DMF. Activated esters were isolated in quantitative yield (~ 95%) by reverse phase chromatography in a neutral solvent mixture (water-acetonitrile).

Further condensation of the active dye derivative of the general formula (B) and AAdUTP (1) was carried out in 0.1 M carbonate-bicarbonate aqueous buffer solution upon cooling. The reaction can be carried out with subsequent replacement of anions, treatment with acids, acid salts with other counterions (or without replacing the anion). The reaction product of the general formula (A) was isolated and purified from impurities in two stages. The first purification was carried out using ion-exchange chromatography on a DEAE-cellulose sorbent in a linear gradient of concentrations from 0 to 0.3 M TEANS (triethylammonium hydrocarbonate buffer solution) in 20% acetonitrile. Then, the conjugate of general formula (A) was further purified by reverse phase chromatography on a C18-RP column in an acetonitrile-0.05M triethylammonium hydrogen carbonate buffer solution (TEANS) in a linear concentration gradient from 0 to 50% acetonitrile a. The use of triethylammonium bicarbonate to create a gradient of ionic strength is due to the fact that it can be easily removed in vacuum at room temperature. Triethylammonium ions inhibit the PCR reaction, therefore, after isolation of the fluorescently-labeled AAdUTP of general formula (A) in the form of a triethylammonium salt, it was converted to another salt form.

Modified nucleotides of the general formula (A) were prepared in different salt forms. The use of sodium perchlorate at the last stage of isolation and purification made it possible to obtain nucleotides of formulas A in the form of their sodium salts. Also, nucleotides of formula A can be obtained in phosphoric acid form, in the form of alkali metal salts and in the form of various ammonium salts, using the corresponding reagents instead of sodium perchlorate at the last stage of purification:

- 0.1 M KCl - to obtain the potassium salt of the nucleotide;

- 0.1 M LiCl - to obtain the lithium salt of the nucleotide;

- 0.1 M TEAA - to obtain the triethylammonium salt of the nucleotide;

- 0.1 M NH ₄ HCO ₃ - to obtain the ammonium salt of the nucleotide.

Fractions containing the target compound of general formula (A) were combined and diluted to a volume of 50 ml with deionized water. The concentration of the modified nucleotide of the general formula (A) was determined based on the molar extinction coefficient of cyanine dyes (B). Absorption was determined in triplicate by diluting the stock solution in MQ so that the optical density did not exceed 0.1.

The structure of the intermediate and target compounds was confirmed by UV, ¹ H-NMR spectroscopy, ³¹ P-spectroscopy and mass spectrometry.

Nucleotides of the general formula (A) modified with zwitterionic dyes are characterized by intense absorption and fluorescence bands and a fluorescence quantum yield of 30-34% in PBS. The absorption and fluorescence maxima of dye-modified nucleotides are located in the region of 650 and 665 nm in the case of nucleoside triphosphates of the general formula A (n = 2) and in the regions of 750 and 765 nm in the case of nucleoside triphosphates of the general formula A (n = 3).

Another object of the invention is the use of the compounds of the invention of general formula (A) for enzymatic fluorescence labeling of DNA during PCR. The efficiency of incorporation of modified nucleotides (A) under PCR conditions was analyzed using the commercial KRAS-biochip test system (Biochip-IMB, Moscow, Russia), designed to determine mutations characteristic of pancreatic cancer (M. A. Emelyanova. Definition mutations in the KRAS gene in tumor cells using biological microarrays // Molecular Biology. - 2011. - T. 45, No. 5. - S. 863-870). In this case, the correctly selected structure of the dye, which is part of the labeled deoxynucleoside triphosphate, can lead to an increase in the sensitivity of molecular genetic analysis. Fluorescence measurements were performed on a Chipdetector-01 instrument (Biochip-IMB, Russia) for analysis of nucleoside triphosphates of the general formula A (n = 2) and on a fluorescence analyzer for the analysis of biochips with IR fluorescent labels for the study of nucleoside triphosphates of the general formula A ( n = 3).

The microchip is a hydrogel cell located on a substrate with oligonucleotide probes immobilized inside the cells. The cell diameter is approximately 100 microns. In different cells are oligonucleotide probes of various structures. In cells in which the structure of the probe corresponds to the structure of the amplicon, the amplicon binds to a durable complex, duplex. In those cells in which the probe structure does not correspond to the amplicon structure, amplicon binding does not occur. The results of the analysis are evaluated by the intensity of the fluorescent signals in the cells of the biochip. In the cells in which the binding of the fluorescently-labeled amplicon to the probe occurred, an intense fluorescence signal is observed. To register signals using a fluorescence microscope equipped with a computer with special software.

Various factors affect the magnitude of the signals: the efficiency of incorporation of modified nucleotides by DNA polymerase into the amplified product, specific binding to form perfect duplexes with oligonucleotide probes located in the biochip cells, non-specific binding to components of the biochip cell, fluorescence brightness of the fluorescence tag associated with the amplification much more.

To assess the degree of incorporation of modified nucleotides with Taq polymerase into DNA, the average fluorescence signal of perfect duplexes in the biochip cells was calculated (Kuznetsova V. E., Spitsyn M. A., Shershov V. E., Guseinov T. O., Fesenko E. E. , Lapa SA, Ikonnikova A. Yu., Avdonina M. A., Nasedkina T. V., Zasedatelev AS, A. V. Chudinov. // Mend. Commyn. 2016. V. 26.P. 95-98; Maksim A. Spitsyn, Viktoriya E. Kuznetsova, Valeriy E. Shershov, Marina A. Emelyanova, Teimur O. Guseinov, Sergey A. Lapa, Tatyana V. Nasedkina, Alexander S. Zasedatelev, Alexander V. Chudinov, Synthetic route to novel zwitterionic pentamethine indocyanine fluorophores with various substitutions // Dyes and Pigments, V. 147, P. 199-210) and then normalized it to the value obtained for a commercially available product (AAdUTP-Cy5) (Jena Bioscience GmbH, Germany, NU-803- CY5-S).

The fluorescent signal was higher for nucleotides A10-A18 containing zwitterionic indotricarbocyanine dye. For both groups of modified nucleoside triphosphates A1-A9 and A10-A18, the dependence of the normalized fluorescence signal on the length of the linker separating the fluorophore from the pyrimidine base, which varies in the series: (A3)> (A2)> (A1); (A6)> (A5)> (A4); (A9)> (A8)> (A7); (A12)> (A11)> (A10); (A15)> (A14)> (A13); (A18)> (A17)> (A16). The fluorescent signals obtained for nucleotides (A) with a long linker containing a dye of the general formula (C) and (D, m = 3) in their structure were significantly higher than the signals obtained using a commercially available AAdUTP-Cy5 containing structure disulfonated and dodicarbocyanine dye.

The use of fluorescence-labeled nucleotides of the general formula (A) in PCR and registration of fluorescence in the infrared region of the spectrum allows to obtain a fluorescent signal 20-25 times higher compared to the commercially available product AAdUTP-Cy5 when determining mutations of pancreatic cancer using test systems KRAS-biochip (Biochip-IMB, Moscow, Russia). Based on the foregoing, it follows that the inventive fluorescently-labeled nucleoside triphosphates of the general formula (A) can be successfully used for enzymatic labeling of DNA during PCR.

The following examples illustrate the invention.

Examples of compounds and their use

In the work we used reagents and solvents of “OSCh”, “ChCh” or “ChDA” brands of Aldrich, Alfa Aesar, Fluka, Himmed firms.

DMF was purified by vacuum distillation in a stream of nitrogen over phthalic anhydride [t. bale. 46 ° C (10 Torr)]. Diisopropylethylamine was distilled over ninhydrin, then over KOH (mp. 126-127 ° C). Acetic anhydride was boiled with anhydrous potassium acetate, and then distilled (t. Boiling. 139-140 ° C).

1 M Triethylammonium acetate buffer solution (TEAA) was prepared as follows. While cooling on ice and vigorous stirring, acetic acid (57 ml, 1 mol) was added dropwise to a mixture of deionized water (700 ml) and triethylamine (139 ml, 1 mol) and the solution volume was adjusted to 1 l

1 M Triethylammonium-bicarbonate buffer solution (TEANS) was prepared as follows. Under cooling on ice, vigorous stirring and pH control, gaseous CO ₂ (to pH 7.5) was passed into a mixture of deionized water (700 ml) and triethylamine (139 ml, 1 mol) and the volume of the solution was adjusted to 1 l.

0.1 M Phosphate-buffered saline (PBS), 0.15 M NaCl, pH 7.4 was prepared as follows. A mixture of disubstituted potassium phosphate (0.87 g, 0.005 mol), monosubstituted potassium phosphate (0.68 g, 0.005 mol) and sodium chloride (9.0 g, 0.15 mol) was dissolved in deionized water (700 ml). It was alkalinized with a 1 M solution of monosubstituted potassium phosphate to pH 7.4 and the volume of the resulting solution was adjusted to 1 L.

Tris / EDTA buffer solution (TE buffer, 10 mM Tris (pH 8.0), 1 mM EDTA) was purchased from Aldrich.

Example 1. Obtaining phenylhydrazine-n-sulfonic acid (compound 6)

Sulfanilic acid (52.0 g, 0.3 mol) and sodium carbonate (16.5 g, 0.06 mol) are dissolved by heating in water (200 ml). The resulting solution was filtered off and concentrated sulfuric acid (35.0 g, 19.1 ml, 0.36 mol) was added dropwise, with a white precipitate. To the suspension, while cooling on ice and stirring, a solution of sodium nitrite (21.0 g, 0.3 mol) in water (50 ml) is added at such a rate that the temperature of the reaction mass does not rise above 12 ° C. Additional stirring was continued for another 15 minutes. The precipitated diazocompound precipitate was collected by filtration and washed with a small amount of ice water.

To a solution of sodium sulfite (85 g, 0.67 mol) in water (250 ml), with stirring and cooling on ice with salt, wet diazocompound is added in portions at such a rate that the temperature of the reaction mass is below + 5 ° C. Stir for 1 hour more. Then the mixture is heated to boiling and concentrated hydrochloric acid (200 ml) is added dropwise, and a precipitate forms. To completely discolor the mixture, zinc dust (2.0 g) is added. The reaction mass was incubated for 12 hours at room temperature. The precipitate was collected by filtration, washed with cold water and dried in air. Phenylhydrazine-n-sulfonic acid (compound 6) is obtained in a yield of 42 g (74%).

Example 2. Obtaining a potassium salt of 2,3,3-trimethyl-5-sulfoindolenine (compound 7)

A mixture of phenylhydrazine-n-sulfonic acid (compound 6) (11.0 g, 0.06 mol), 3-methyl-2-butanone (7.7 ml, 0.072 mol) and glacial acetic acid (34 ml) was boiled for 3 hours, and then held for 12 h at 0 ° C. The precipitate was separated by filtration, washed with acetone and dried in a vacuum desiccator over P ₂ O ₅ . Obtain 5.1 g (71%) of indolenin.

¹ H NMR (D ₂ O, δ, ppm): 1.28 (s, 6H, 2C (3) H ₃ ), 7.47-7.79 (3H, m, Ar H )

5-Sulfo-2,3,3-trimethylindolenine (4.8 g, 0.02 mol) was dissolved by heating in methanol (25 ml). To the resulting solution was added dropwise a solution of potassium hydroxide (1.4 g, 0.024 mol) in methanol (16 ml). Isopropyl alcohol (50 ml) is then added. The precipitate is filtered off, dried in a vacuum desiccator over P ₂ O ₅ . Obtain 4.7 g (85%) of indolenin (compound 7) in the form of brilliant plates of light beige color. λ _max 260 nm. Mass spectrum (MALDI) (C ₁₁ H ₁₂ NO ₃ S ^- ): found m / z 239.0, calculated m / z 238.28.

¹ H NMR (D ₂ O, δ, ppm): 1.19 (6H, s, 2C (3) H ₃ ), 2.19 (3H, s, C (2) H ₃ ), 7.41-7.7 (3H, m, Ar H )

Example 3. Obtaining 2,3,3-trimethyl-5-sulfo-N - ((3-trimethylammonium) propyl) indoleninium bromide (compound 2).

A mixture of the potassium salt of 2,3,3-trimethyl-5-sulfoindolenine (compound 7) (0.35 g, 1.26 mmol), (3-bromopropyl) trimethyl ammonium bromide (0.4 g, 1.39 mmol) and butyronitrile (5 ml) is heated in nitrogen atmosphere for 72 hours at 75 ° C. At the end of heating, acetone (30 ml) was added. The resulting crystals are filtered off, recrystallized from methanol and dried in a vacuum desiccator over P ₂ O ₅ . Obtain 0.4 g (86%) of indoleninium salt (compound 2), so pl. 248 ° C. λ _max 228 nm.

¹ H NMR (DMSO-d6) δ: 8.03, 7.79 (2 m, 3H, ArH), 4.50 (m, 2H, C (1) H ₂ ), 3.66 (m, 2H, C (3) H ₂ ), 3.14 [s, 9H, N (CH ₃ ) ₃ ], 2.34 (m, 2H, C (2) H ₂ ), 1.57 [s, 6H, 2CH ₃ (3)]. Mass spectrum (MALDI) (C ₁₇ H ₂₇ N ₂ O ₃ S ⁺ ): found m / z 340.2 [M] ⁺ , calculated m / z 339.47.

Example 4. Obtaining 2,3,3-trimethyl-5-sulfo-N - ((5-trimethylammonium) pentyl) indoleninium bromide (compound 2).

Compound 2 was obtained analogously to compound 1. Obtain 0.46 mg (81%) of compound 3, so pl. 250 ° C. λ _max 228 nm.

¹ H NMR (DMSO-d6) δ: 8.03, 7.97, 7.84 (3m, 3H, ArH), 4.52 (m, 2H, C (1) H ₂ ), 3.34 (m, 2H, C (5) H ₂ ) 3.08 [s, 9H, N (CH ₃ ) ₃ ], 1.92 (m, 2H, C (4) H ₂ ), 1.75 (m, 2H, C (3) H ₂ ), 1.56 [s, 6H, 2CH ₃ (3)], 1.35 (m, 2H, C (2) H ₂ ). Mass spectrum (MALDI) (C ₁₉ H ₃₁ N ₂ O ₃ S ⁺ ): found m / z 368.3 [M] ⁺ , calculated m / z 367.53.

Example 5. Obtaining 2,3,3-trimethyl-5-sulfo-N-ethylindoleninium (compound 4)

A mixture of the potassium salt of 2,3,3-trimethyl-5-sulfoindolenine (compound 7) (1.1 g, 4 mmol), 6-bromohexanoic acid (2.0 g, 10 mmol) and 1,2-dichlorobenzene was heated for 30 h at 145 ° С in an atmosphere of nitrogen. At the end of heating, the mixture was cooled to room temperature, the solvent was decanted, and the residue was triturated with diethyl ether. The resulting crystals are filtered off, washed thoroughly with acetone and dried in a vacuum desiccator over P ₂ O ₅ . The product yield (compound 4) was 1 g (75%). UV spectrum (MeOH), λ _max / nm: 202, 275 nm. Found: m / z 353.9 [M] ⁺ . C ₁₇ H ₂₃ NO ₅ S. Calculated: M = 353.43.

¹ H NMR (D ₂ O, δ, ppm, J / Hz): 1.31, 1.49, 1.7, 2.11 (m, 8H, CH ₂ C H ₂ C H ₂ C H ₂ C H ₂ COOH), 1.4 (s, 6H, 2C (3) H ₃ ), 4.34 (m, 2H, C H ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH), 7.6 (d, 1H, ArH, J = 8.5), 7.73 (d, 1H, ArH, J = 8.5), 7.96 (s, 1H, Ar H )

Example 6. Obtaining 1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -1- (5-carboxypentyl) -3,3,3', 3-tetramethyl-5,5'-disulfoindodicarbocyanine dye (compound C1).

A mixture of N- (5-carboxypentyl) -2,3,3-trimethyl-5-sulfoindoleninium (compound 4) (177 mg, 0.5 mmol), malonic dialdehyde dianyl hydrochloride (compound 3) (78 mg, 0.3 mmol), acetic anhydride (3 ml), acetic acid (1 ml) and anhydrous potassium acetate (196 mg, 2 mmol) are heated for 5 hours at + 118 ° C. The solvents are removed, 3 ml of 0.5 M HCl are added to the residue and heated for 4 hours at 90 ° C. At the end of heating, water (50 ml) was added and the reaction mass was neutralized with a NaHCO ₃ solution to pH 7. The reaction products were isolated by reverse phase chromatography on an RP-18 column in an acetonitrile-0.1 M TEAA gradient elution from 0 to 50% acetonitrile. The solvents are removed, and the residue is diluted with water, again applied to an RP-18 column, washed with 0.1 M NaCl solution, water, then isolated by reverse phase chromatography in an acetonitrile-water system. Obtained 100 mg (54%) of the sodium salt of N, N'-di- (5-carboxypentyl) ^-3,3,3, 3'-tetramethyl-5,5'-disulfoindodikarbotsianinovogo dye (Compound 5, n = 2). Found: m / z 743.6 [M] ⁺ . C ₃₇ H ₄₅ N ₂ O ₁₀ S ₂ ^- . Calculated: M = 741.89.

¹ H NMR (DMSO-d ₆ , δ, ppm, J / Hz): 1.37, 1.54, 1.75, 2.16 (4 m, 16H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH), 1.69 (s , 12H, C (3.3) H ₃ , C (3 ', 3') H ₃ ), 4.09 (m, 4H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH), 6.31 (m, 2H, α , α'-CH), 6.60 (t, 1H, γ-CH, J = 12.0), 7.8, 7.63, 7.32 (3m, 6H, ArH), 8.35 (t, 2H, β, β'-CH, J = 13.0)

A mixture of the sodium salt of N, N'-di- (5-carboxypentyl) -3,3,3 ', 3'-tetramethyl-5,5'-disulfoindodicarbocyanine dye (compound 5) (36 mg, 48 μmol), HBTU (74 mg, 0.19 mmol), N-hydroxysuccinimide (29 mg, 0.24 mmol) was dissolved in DMF (1 ml) and stirred for 2 h at ~ 20 ° С. Then, a solution of 3-dimethylaminopropylamine (8.5 mg, 48 μmol) in a carbonate-bicarbonate buffer solution (1 ml, 0.1 M, pH 9.2) is introduced into the reaction mass with stirring. The reaction mass is stirred for 24 hours at ~ 20 ° C, then diluted with 0.1 M TEAA (5 ml). The target compound is isolated analogously to compound 5. Receive 20 mg (50%) of a modified dye (compound 6).

¹ H NMR (DMSO-d ₆ ) δ: 8.35 (t, 2H, β, β'-CH, J 13.0 Hz), 7.81, 7.65, 7.31 (3m, 6H, ArH), 6.59 (t, 1H, γ- CH, J 12.0 Hz), 6.31 (m, 2H, α, α'-CH), 4.09 (m, 4H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH, C (1) H ₂ ), 3.01 (m , 2H, C (8) H ₂ ), 2.41 (m, 2H, C (10) H ₂ ), 2.29 [s, 6H, N (CH ₃ ) ₂ ], 2.03 (m, 2H, C (9) H ₂ ), 1.69 [s, 12H, C (3.3) H ₃ , C (3.3 ') H ₃ ], 2.19, 1.75, 1.53, 1.35 (4 m, 16H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH, C (2) H ₂ , C (3) H ₂ , C (4) H ₂ , C (5) H ₂ ). Found: m / z 828.1 [M] ⁺ . C ₄₂ H ₅₇ N ₄ O ₉ S ₂ ^- . Calculated: M = 826.06.

A mixture of the modified dye (compound 6) (20 mg, 24 μmol), methyl iodide (300 μl), DMF (2 ml) and DIPEA (50 μl) was stirred for 1 h at ~ 20 ° C. The solvents were removed in vacuo, the residue was dissolved in 0.5M HCl (4 ml) and heated for 1 h at 85 ° C. The reaction mass is diluted with 0.1 M TEAA. The reaction products are isolated by reverse phase chromatography on an RP-18 column in an acetonitrile-0.1 M TEAA system, gradient eluting from 0 to 50% acetonitrile. The solvents are removed, and the residue is diluted with water, again applied to an RP-18 column, washed with 0.1 M NaCl solution, water, then isolated by reverse phase chromatography in an acetonitrile-water system. Received 10 mg (50%) of compound C1.

¹ H NMR (DMSO-d ₆ ) δ: 8.35 (t, 2H, β, β'-CH, J13.0 Hz), 7.82, 7.63, 7.31 (3m, 6H, ArH), 6.58 (t, 1H, γ -CH, J 12.0 Hz), 6.32 (m, 2H, α, α'-CH), 4.09 (m, 4H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH, C (1) H ₂ ), 3.23 ( m, 2H, C (8) H ₂ ), 3.01 [s, 9H, N (CH ₃ ) ₃ ], 2.97 (m, 2H, C (10) H ₂ ), 2.04 (m, 2H, C (9) H ₂ ), 1.69 [s, 12H, C (3.3) H ₃ , C (3.3 ') H ₃ ], 2.2, 1.75, 1.53, 1.35 (4 m, 16H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH, C (2) H ₂ , C (3) H ₂ , C (4) H ₂ , C (5) H ₂ ). Found: m / z 842.5 [M] ⁺ . C ₄₃ H ₆₀ N ₄ O ₉ S ₂ . Calculated: M = 841.09.

Example 7. Obtaining N- (7-aza-12-carboxy-6-oxododec-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (7-aza-10-trimethylammonio-6- oxodec-1-yl) -5,5'-disulfoindodicarbocyanine dye (compound C2).

A mixture of N '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3', 3'-tetramethyl-N- (5-carboxypentyl) -5,5'-disulfoindodicarbocyanine dye ( compound C1) (11 mg, 13 μmol), p-nitrophenol (4 mg, 30 μmol), HBTU (7.5 mg, 20 μmol) are dissolved in a mixture of DMF (0.3 ml) and DIPEA (10 μl) and stirred for 2 hours at ~ 20 ° C. Then the reaction mass is cooled to -18 ° C and a solution of 6-aminocaproic acid (34 mg, 0.26 mmol) in 0.1 M carbonate-bicarbonate buffer solution (0.3 ml) is introduced with stirring. The reaction mass was incubated for 3 hours at ~ 20 ° C, then diluted with 0.1 M TEAA. The target compound is isolated analogously to compound C1. Obtain 11 mg (89%) of compound C2.

¹ H NMR (DMSO-d ₆ ) δ: 8.35 (m, 2H, β, β'-CH), 7.82, 7.66, 7.31 (3m, 6H, ArH), 6.58 (t, 1H, γ-CH, J 12.0 Hz), 6.31 (m, 2H, α, α'-CH, J 13.5 Hz), 4.10 (m, 4H, C (1) H ₂ , C (1 ') H ₂ ), 3.23 (m, 4H, C (8) H ₂ , C (8 ') H ₂ ), 3.01 [s, 11H, N (CH ₃ ) ₃ , C (10') H ₂ ], 2.46 (m, 4H, C (5) H ₂ , C (5 ') H ₂ ), 2.17 (m, 2H, C (12) H ₂ ), 2.03 (m, 4H, C (9) H ₂ , C (9') H ₂ ), 1.69 [s, 12H , C (3.3) H ₃ , C (3.3 ') H ₃ ], 1.76, 1.54, 1.33 (3 br.m., 16H, C (2) H ₂ , C (3) H ₂ , C (4) H ₂ , C (10) H ₂ , C (11) H ₂ , C (2 ') H ₂ , C (3') H ₂ , C (4 ') H ₂ ). Found: m / z 955.7 [M] ⁺ . C ₄₉ H ₇₁ N ₅ O ₁₀ S ₂ . Calculated: M = 954.25.

Example 8. Obtaining N- (7,14-diaza-19-carboxy-6,13-dioxononadec-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (7-aza-10- trimethylammonio-6-oxodec-1-yl) -5,5'-disulfoindodicarbocyanine dye (compound C3).

Mixture of N- (7-aza-12-carboxy-6-oxododecan-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (1-aza-10-trimethylammonio-6-oxodecane-1 -yl) -5,5'-disulfoindodicarbocyanine dye (compound C2) (6 mg, 6 μmol), p-nitrophenol (3 mg, 20 μmol), HBTU (4 mg, 10 μmol) are dissolved in a mixture of DMF (0.2 ml) and DIPEA (5 μl) and stirred for 3 hours at ~ 20 ° C. Then the reaction mass is cooled to -18 ° C and a solution of 6-aminocaproic acid (16 mg, 0.12 mmol) in 0.1 M carbonate-bicarbonate buffer solution (0.2 ml) is introduced with stirring. The reaction mass is kept for 2 hours at ~ 20 ° C, then diluted with 0.1 M TEAA. The target compound is isolated analogously to compound C1. 5 mg (78%) of compound C3 are obtained.

¹ H NMR (DMSO-d ₆ ) δ: 8.32 (m, 2H, β, β'-CH), 7.83, 7.62, 7.35 (3m, 6H, ArH), 6.56 (t, 1H, γ-CH, J 12.0 Hz), 6.35 (d, 2H, α, α'-CH, J 13.5 Hz), 4.08 [m, 4H, C (1) H ₂ , C (1 ') H ₂ ], 3.25 [m, 6H, C (8) H ₂ , C (15) H ₂ , C (8 ') H ₂ ], 3.04 [s, 11H, N (CH ₃ ) ₃ , C (10') H ₂ ], 2.48 [m, 6H, C (5) H ₂ , C (12) H ₂ , C (5 ') H ₂ ], 2.15 [m, 2H, C (19) H ₂ ], 2.05 [m, 6H, C (9) H ₂ , C (16) H ₂ , C (9 ') H ₂ ], 1.71 [s, 12H, C (3.3) H ₃ , C (3.3') H ₃ ], 1.79, 1.52, 1.31 [3 ears . m., 20H, C (2) H ₂ , C (3) H ₂ , C (4) H ₂ , C (10) H ₂ , C (11) H ₂ , C (17) H ₂ , C (18 ) H ₂ , C (2 ') H ₂ , C (3') H ₂ , C (4 ') H ₂ ]. Found: m / z 1068.9 [M] ⁺ . C ₅₅ H ₈₂ N ₆ O ₁₁ S ₂ . Calculated: M = 1067.41.

Example 9. Obtaining N- (5-carboxypentyl) -3,3,3 ', 3'-tetramethyl-N' - (3-trimethylammoniopropyl) -5,5'-disulfoindodicarbocyanine dye (compound D1).

A mixture of N- (5-carboxypentyl) -2,3,3-trimethyl-5-sulfoindoleninium (compound 4) (124 mg, 0.35 mmol), malonic dialdehyde dianyl hydrochloride (compound 3) (78 mg, 0.3 mmol), acetic anhydride (1.5 ml), acetic acid (500 μl) is heated for 2 hours at 118 ° C. At the end of heating, 2,3,3-trimethyl-5-sulfo-N- (3-trimethylammoniumpropyl) indolenium bromide (compound 2) (210 mg, 0.5 mmol), anhydrous potassium acetate (170 mg, 1.75 mmol) are added to the reaction mass. ), acetic anhydride (2 ml), acetic acid (1 ml) and heated for 2 hours at 118 ° C. Compound D1 is isolated analogously to compound C1. 100 mg (46%) of compound D1 is obtained.

¹ H NMR (DMSO-d ₆ ) δ: 8.36 (m, 2H, β, β'-CH), 7.80, 7.61, 7.32 (3m, 6H, ArH), 6.59 (t, 1H, γ-CH, J 12.0 Hz), 6.29 (m, 2H, α, α'-CH), 4.05 (m, 4H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ , CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH), 3.49 [m, 2H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ], 3.04 [s, 9H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ], 2.09 [m, 2H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ], 1.65 [s, 12H, CH ₃ (3.3), CH ₃ (3.3 ')], 1.96, 1.47, 1.31 (3m, 8H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH). MS (MALDI-TOF) m / z: calcd. for C ₃₇ H ₄₉ N ₃ O ₈ S ₂ 727.93; found 729.1 [M] ⁺ .

Example 10. Obtaining N- (7-aza-12-carboxy-6-oxododec-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (3-trimethylammoniopropyl) -5,5'- disulfoindodicarbocyanine dye (compound D2).

A mixture of N- (5-carboxypentyl) -3,3,3 ', 3'-tetramethyl-N' - (3-trimethylammoniumpropyl) -5,5'-disulfoindodicarbocyanine dye (compound D1) (12 mg, 17.7 μmol), p -nitrophenol (6 mg, 42 μmol), HBTU (9 mg, 24 μmol) are dissolved in a mixture of DMF (0.3 ml) and DIPEA (15 μl) and stirred for 3 hours at ~ 20 ° С. Then the reaction mass is cooled to -18 ° C and a solution of 6-aminocaproic acid (46 mg, 0.35 mmol) in 0.1 M carbonate-bicarbonate buffer solution (0.3 ml) is introduced with stirring. The reaction mass was incubated for 12 h at ~ 20 ° C, then diluted with 0.1 M TEAA. The target compound is isolated analogously to compound C1. Obtain 11 mg (70%) of compound D2.

¹ H NMR (DMSO-d6) δ: 8.41 (m, 2H, β, β'-CH), 7.86, 7.66, 7.35 (3m, 6H, ArH), 6.59 (t, 1H, γ-CH, J 12.0 Hz ), 6.33 (m, 2H, α, α'-CH), 4.13 (m, 4H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ , C (1) H ₂ ), 3.45 [m, 2H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ], 3.08 [s, 9H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ], 2.98 [m, 2H, C (8) H ₂ ], 2.44 [m , 2H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ], 1.71 [s, 12H, CH ₃ (3.3), CH ₃ (3.3 ')], 2.16, 2.03, 1.50, 1.33, 1.22 (5m, 16H, C (2) H ₂ , C (3) H ₂ , C (4) H ₂ , C (5) H ₂ , C (9) H ₂ , C (10) H ₂ , C (11 ) H ₂ , C (12) H ₂ ). Found: m / z 842.5 [M] ⁺ . C ₄₃ H ₆₀ N ₄ O ₉ S ₂ . Calculated: M = 841.09.

Example 11. Obtaining N- (7,14-diaza-19-carboxy-6,13-dioxononadec-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (3-trimethylammoniopropyl) -5 , 5'-disulfoindodicarbocyanine dye (compound D3).

A mixture of N-7-aza-12-carboxy-6-oxododecan-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (3-trimethylammonium propyl) -5,5'-disulfoindodicarbocyanine dye (compound D2) (10 mg, 11 μmol), p-nitrophenol (5 mg, 33 μmol), HBTU (6 mg, 15 μmol) are dissolved in a mixture of DMF (0.3 ml) and DIPEA (5 μl) and stirred for 3 h at ~ 20 ° C. Then the reaction mass is cooled to -18 ° C and a solution of 6-aminocaproic acid (29 mg, 0.22 mmol) in 0.1 M carbonate-bicarbonate buffer solution (0.3 ml) is introduced with stirring. The reaction mass was incubated for 12 h at ~ 20 ° C, then diluted with 0.1 M TEAA. The target compound is isolated analogously to compound C1. 7.5 mg (76%) of compound D3 are obtained.

¹ H NMR (DMSO-d ₆ ) δ: 8.39 (m, 2H, β, β'-CH), 7.77, 7.59, 7.32 (3m, 6H, ArH), 6.58 (t, 1H, γ-CH, J 12.0 Hz), 6.31 (m, 2H, α, α'-CH), 4.08 (m, 4H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ , C (1) H ₂ ), 3.50 [m, 2H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ], 3.05 [s, 9H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ], 2.93 [m, 4H, C (8) H ₂ , C (15 ) H ₂ ], 2.48 [m, 2H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ], 1.67 [s, 12H, CH ₃ (3.3), CH ₃ (3.3 ')], 2.09 , 2.0, 1.85, 1.45, 1.31, 1.15 (5m, 16H, C (2) H ₂ , C (3) H ₂ , C (4) H ₂ , C (5) H ₂ , C (9) H ₂ , C (10) H ₂ , C (11) H ₂ , C (12) H ₂ , C (16) H ₂ , C (17) H ₂ , C (18) H ₂ , C (19) H ₂ ). Found: m / z 955.6 [M] ⁺ . C ₄₉ H ₇₁ N ₅ O ₁₀ S ₂ . Calculated: M = 954.25.

Example 12. Obtaining N '- (5-carboxypentyl) -3,3,3', 3'-tetramethyl-N '- (5-trimethylammoniopentyl) -5,5'-disulfoindodicarbocyanine dye (compound D4).

N- (5-Carboxypentyl) -3,3,3 ', 3'-tetramethyl-N' - (3-trimethylammonium pentyl) -5,5'-disulfoindodicarbocyanine dye (compound D4) is obtained analogously to compound D1 in the following reagent ratios: ( Stage 1) N- (5-carboxypentyl) -2,3,3-trimethyl-5-sulfoindoleninium (compound 4) (124 mg, 0.35 mmol), malonic dialdehyde dianyl hydrochloride (compound 3) (78 mg, 0.3 mmol), acetic anhydride (1.5 ml), acetic acid (0.5 ml); (Stage 2) 2,3,3-trimethyl-5-sulfo-N- (5-trimethylammonium pentyl) indolenium bromide (compound 2) (224 mg, 0.5 mmol), anhydrous potassium acetate (170 mg, 1.75 mmol), acetic anhydride (2 ml), acetic acid (1 ml). 184 mg (81%) of compound D4 is obtained.

¹ H NMR (DMSO-d ₆ ) δ: 8.36 (m, 2H, β, β'-CH), 7.83, 7.64, 7.34 (3m, 6H, ArH), 6.58 (t, 1H, γ-CH, J 12.0 Hz), 6.32 (m, 2H, α, α'-CH), 4.12 (m, 4H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ , CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH), 3.21 [m, 2H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ], 3.01 [s, 9H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ] , 1.69 [s, 12H, CH ₃ (3.3), CH ₃ (3.3 ')], 2.19, 1.76, 1.51, 1.35 [4m, 14H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ]. Found: m / z 757.1 [M] +. C ₃₉ H ₅₃ N ₃ O ₈ S ₂ . Calculated: M = 755.99.

Example 13. Obtaining N- (7-aza-12-carboxy-6-oxododec-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (3-trimethylammoniopentyl) -5,5'- disulfoindodicarbocyanine dye (compound D5).

A mixture of N- (5-carboxypentyl) -3,3,3 ', 3'-tetramethyl-N' - (3-trimethylammoniumpropyl) -5,5'-disulfoindodicarbocyanine dye (compound D4) (20 mg, 26 μmol), p -nitrophenol (8 mg, 56 μmol), HBTU (13 mg, 35 μmol) are dissolved in a mixture of DMF (0.7 ml) and DIPEA (15 μl) and stirred for 4 hours at ~ 20 ° С. Then the reaction mass is cooled to -18 ° C and a solution of 6-aminocaproic acid (68 mg, 0.52 mmol) in 0.1 M carbonate-bicarbonate buffer solution (0.7 ml) is introduced with stirring. The reaction mass was incubated for 12 h at ~ 20 ° C, then diluted with 0.1 M TEAA. The target compound is isolated analogously to compound C1. 19 mg (83%) of compound D5 are obtained.

'H NMR (DMSO-d ₆ ) δ: 8.38 (m, 2H, β, β'-CH), 7.84, 7.62, 7.33 (3m, 6H, ArH), 6.57 (t, 1H, γ-CH, J 12.0 Hz), 6.31 (m, 2H, α, α'-CH), 4.11 (m, 4H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ , C (1) H ₂ ), 3.25 [ m, 2H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ], 3.04 [s, 9H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ], 2.95 [m, 2H, C (8) H ₂ ], 1.68 [s, 12H, CH ₃ (3.3), CH ₃ (3.3 ')], 2.46, 2.18, 1.52, 1.33, 1.22 [4 m, 22H, C (2) H ₂ , C (3) H ₂ , C (4) H ₂ , C (5) H ₂ , C (9) H ₂ , C (10) H ₂ , C (11) H ₂ , C ( 12) H ₂ , CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ]. Found: m / z 870.3 [M] ⁺ . C ₄₅ H ₆₄ N ₄ O ₉ S ₂ . Calculated: M = 869.14.

Example 14. Obtaining N- (7,14-diaza-19-carboxy-6,13-dioxononadec-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (3-trimethylammoniopentyl) -5 , 5'-disulfoindodicarbocyanine dye (compound D6).

A mixture of N- (7-aza-12-carboxy-6-oxododec-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (3-trimethylammoniumpropyl) -5,5'-disulfoindodicarbocyanine dye ( compound D5) (6 mg, 6.7 μmol), p-nitrophenol (3 mg, 20 μmol), HBTU (6 mg, 15 μmol) are dissolved in a mixture of DMF (0.3 ml) and DIPEA (10 μl) and stirred for 3 hours at ~ 20 ° C. Then the reaction mass is cooled to -18 ° C and a solution of 6-aminocaproic acid (18 mg, 0.13 mmol) in 0.1 M carbonate-bicarbonate buffer solution (0.3 ml) is introduced with stirring. The reaction mass was incubated for 12 h at ~ 20 ° C, then diluted with 0.1 M TEAA. The target compound is isolated analogously to compound C1. 5 mg (77%) of compound D6 are obtained.

¹ H NMR (DMSO-d ₆ ) δ: 8.39 (m, 2H, β, β'-CH), 7.81, 7.62, 7.31 (3m, 6H, ArH), 6.58 (t, 1H, γ-CH, J 12.0 Hz), 6.32 (m, 2H, α, α'-CH), 4.11 (m, 4H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ , C (1) H ₂ ), 3.25 [ m, 2H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ], 3.02 [s, 9H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ], 2.95 [m, 4H, C (8) H ₂ , C (15) H ₂ ], 1.72 [s, 12H, CH ₃ (3.3), CH ₃ (3.3 ')], 2.48, 2.11, 2.02, 1.81, 1.42 , 1.34, 1.17 [7m, 30H, C (2) H ₂ , C (3) H ₂ , C (4) H ₂ , C (5) H ₂ , C (9) H ₂ , C (10) H ₂ , C (11) H ₂ , C (12) H ₂ , C (16) H ₂ , C (17) H ₂ , C (18) H ₂ , C (19) H ₂ , CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ]. Found: m / z 983.2 [M] ⁺ . C ₅₁ H ₇₅ N ₅ O ₁₀ S ₂ . Calculated: M = 982.30.

Example 15. Obtaining 1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -1- (5-carboxypentyl) -3,3,3', 3'-tetramethyl-5,5'- disulfoindotricarbocyanine dye (compound C4).

A mixture of N- (5-carboxypentyl) -2,3,3-trimethyl-5-sulfoindoleninium (compound 4) (71 mg, 0.2 mmol), glutacone dialdehyde dianyl hydrochloride (compound 3) (86 mg, 0.3 mmol), acetic anhydride (2 ml) and acetic acid (0.7 ml) are heated for 3 hours at 118 ° C. At the end of heating, N- (5-carboxypentyl) -2,3,3-trimethyl-5-sulfoindoleninium (compound 4) (107 mg, 0.3 mmol), acetic anhydride (2 ml), acetic acid (1 ml) are added to the reaction mass. ), anhydrous potassium acetate (200 mg, 2 mmol) and heated for 5 h at 80 ° C. The solvent is removed, and the reaction product is isolated by reverse phase chromatography on an RP-18 column in an acetonitrile-0.1 M TEAA gradient gradient elution from 0 to 50% acetonitrile. The solvents are removed, and the residue is diluted with water, again applied to an RP-18 column, washed with 0.1 M NaCl solution, water, then isolated by reverse phase chromatography in an acetonitrile-water system. Received 68 mg (43%) of compound 5 (n = 3).

¹ H NMR (DMSO-d ₆ ) δ: 7.86 (t, 2H, β, β'-CH, J 12.0 Hz), 7.71 (m, 1H, δ-CH), 7.17-7.52 (m, 6H, ArH) 6.54 (t, 2H, γ, γ'-CH, J 12.0 Hz), 6.32 (m, 2H, α, α'-CH), 4.1 (m, 4H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH ), 1.63 [s, 12H, CH ₃ (3.3), CH ₃ (3.3 ')], 2.13, 1.74, 1.52, 1.36 (4m, 16H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH) . Found: m / z 161A [M] ⁺ . C ₃₉ H ₄₇ N ₂ O ₁₀ S ₂ ^- . Calculated: M = 767.93.

A mixture of the sodium salt of N, N'-di- (5-carboxypentyl) -3,3,3 ', 3'-tetramethyl-5,5'-disulfoindotricarbocyanine dye (31 mg, 41 μmol), HBTU (61 mg, 0.16 mmol ), N-hydroxysuccinimide (29 mg, 0.24 mmol) was dissolved in DMF (0.6 ml) and stirred for 2 h at ~ 20 ° С. Then, a solution of 3-dimethylaminopropylamine (14 mg, 82 μmol) in a carbonate-bicarbonate buffer solution (0.5 ml, 0.1 M, pH 9.2) is introduced into the reaction mass with stirring. The reaction mass is stirred for 24 hours at ~ 20 ° C, then diluted with 0.1 M TEAA (5 ml). The target compound is isolated analogously to compound 5. Receive 16 mg (44%) of the modified dye 6.

¹ H NMR (DMSO-d ₆ ) δ: 1.3, 1.58, 1.68, 1.78, 2.03 (5m, 18H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH, C (2) H ₂ , C (3) H ₂ , C (4) H ₂ , C (5) H ₂ , C (9) H ₂ ), 1.63 [s, 12H, C (3.3) H ₃ , C (3 ', 3') H ₃ ], 2.24 (s, 6H, N (CH ₃ ) ₂ ), 2.41 (m, 2H, C (10) H ₂ ), 3.01 (m, 2H, C (8) H ₂ ), 4.08 (m, 4H, C ( 1) H ₂ , CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH), 6.34 (m, 2H, α, α'-CH), 6.52 (m, 2H, γ, γ'-CH), 7.29, 7.63, 7.74 (3m, 6H, ArH); 7.87 (m, 3H, δ-CH, β, β'-CH). Found: m / z 854.9 1 [M] ⁺ . C ₄₄ H ₅₉ N ₄ O ₉ S ₂ ^- . Calculated: M = 852.09.

A mixture of the modified dye (30 mg, 34 μmol), methyl p-toluenesulfonate (19 mg, 0.1 mmol) and DMF (1.4 ml) was stirred for 3 h at ~ 20 ° С. The reaction mass is diluted with 0.1 M TEAA and isolated similarly to compound C1. Received 22 mg (73%) of compound C4.

¹ H NMR (DMSO-d ₆ ) δ: 1.35, 1.53, 1.68, 1.75, 2.03 (5m, 16H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH, C (2) H ₂ , C (3) H ₂ , C (4) H ₂ , C (5) H ₂ ), 1.63 [s, 12H, C (3.3) H ₃ , C (3 ', 3') H ₃ ], 3.02 [s, 10H, C (10) H ₂ , N (CH ₃ ) ₃ ], 3.25 (m, 4H, C (8) H ₂ , C (9) H ₂ ), 4.07 (m, 4H, C (1) H ₂ , CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH), 6.38 (m, 2H, α, α'-CH), 6.51 (m, 2H, γ, γ'-CH), 7.27, 7.65, 7.78 (3m, 6H, ArH ), 7.85 (m, 3H, δ-CH, β, β'-CH). Found: m / z 868.5 [M] ⁺ . C ₄₅ H ₆₂ N ₄ O ₉ S ₂ . Calculated: M = 867.13.

Example 16. Obtaining N- (7-aza-12-carboxy-6-oxododec-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (7-aza-10-trimethylammonio-6- oxodec-1-yl) -5,5'-disulfoindotricarbocyanine dye (compound C5).

A mixture of 1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -1 - (5-carboxypentyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindotricarbocyanine dye ( compound C4) (22 mg, 25 μmol) and bis-p-nitrophenyl carbonate (13 mg, 43 μmol) were dissolved in DMSO (0.35 ml) and stirred for 1 h at ~ 20 ° C. Then the reaction mass is cooled to -18 ° C and a solution of 6-aminocaproic acid (34 mg, 0.26 mmol) in 0.1 M carbonate-bicarbonate buffer solution (0.35 ml) is introduced with stirring. The reaction mass is kept for 2 hours at ~ + 5 ° C, then diluted with 0.1 M TEAA. The target compound is isolated analogously to compound C1. 19 mg (89%) of compound C5 are obtained.

¹ H NMR (DMSO-d ₆ ) δ: 7.82 (m, 3H, δ-CH, β, β'-CH), 7.21, 7.62, 7.74 (3m, 6H, ArH), 6.55 (m, 2H, γ, γ'-CH), 6.34 (m, 2H, α, α'-CH), 4.09 (m, 4H, C (1) H ₂ , C (1 ') H ₂ ), 3.25 (m, 4H, C ( 8 ') H ₂ , C (9') H ₂ ), 3.02 [s, 10H, C (10 ') H ₂ , N (CH ₃ ) ₃ ], 2.95 [m, 2H, C (8) H ₂ ] , 1.65 [s, 12H, C (3.3) H ₃ , C (3 ', 3') H ₃ ], 1.32, 1.51, 1.69, 1.78, 2.08 (5m, 24H, C (2) H ₂ , C (3) H ₂ , C (4) H ₂ , C (5) H ₂ , C (9) H ₂ , C (10) H ₂ , C (11) H ₂ , C (12) H ₂ , C ( 2 ') H ₂ , C (3') H ₂ , C (4 ') H ₂ , C (5') H ₂ ). Found: m / z 981.4 [M] ⁺ . C ₅₁ H ₇₃ N ₅ O ₁₀ S ₂ . Calculated: M = 980.29.

Example 17. Obtaining N- (7,14-diaza-19-carboxy-6,13-dioxononadec-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (7-aza-10- trimethylammonio-6-oxodec-1-yl) -5,5'-disulfoindotricarbocyanine dye (compound C6).

Mixture of N- (7-aza-12-carboxy-6-oxododec-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (7-aza-10-trimethylammonio-6-oxodec-1 -yl) -5,5'-disulfoindotricarbocyanine dye (compound C5) (25 mg, 25 μmol) and bis-p-nitrophenyl carbonate (13 mg, 43 μmol) are dissolved in DMSO (0.35 ml) and stirred for 1 h at ~ 20 ° FROM. Then the reaction mass is cooled to -18 ° C and a solution of 6-aminocaproic acid (34 mg, 0.26 mmol) in 0.1 M carbonate-bicarbonate buffer solution (0.35 ml) is introduced with stirring. The reaction mass is kept for 2 hours at ~ + 5 ° C, then diluted with 0.1 M TEAA. The target compound is isolated analogously to compound C1. 19 mg (68%) of compound C6 are obtained.

¹ H NMR (DMSO-d6) δ: 7.87 (m, 3H, δ-CH, β, β'-CH), 7.17, 7.57, 7.71 (3m, 6H, ArH), 6.59 (m, 2H, γ, γ '-CH), 6.32 (m, 2H, α, α'-CH), 4.03 (m, 4H, C (1) H ₂ , C (1') H ₂ ), 3.21 (m, 4H, C (8 ') H ₂ , C (9') H ₂ ), 3.05 [s, 10H, C (10 ') H ₂ , N (CH ₃ ) ₃ ], 2.97 [m, 2H, C (8) H ₂ , C (15) H ₂ ], 1.61 [s, 12H, C (3.3) H ₃ , C (3 ', 3') H ₃ ], 1.35, 1.54, 1.65, 1.74, 2.14 (5 br.m. 32H, C (2) H ₂ , C (3) H ₂ , C (4) H ₂ , C (5) H ₂ , C (9) H ₂ , C (10) H ₂ , C (11) H ₂ , C (12) H ₂ , C (16) H ₂ , C (17) H ₂ , C (18) H ₂ , C (19) H ₂ , C (2 ') H ₂ , C (3') H ₂ , C (4 ') H ₂ , C (5') H ₂ ). Found: m / z 1094.6 [M] ⁺ . C ₅₇ H ₈₄ N ₆ O ₁₁ S ₂ . Calculated: M = 1093.44.

Example 18. Obtaining N- (5-carboxypentyl) -3,3,3 ', 3'-tetramethyl-N' - (3-trimethylammoniopropyl) -5,5'-disulfoindotricarbocyanine dye (compound D7).

A mixture of 2,3,3-trimethyl-5-sulfo-N - ((3-trimethylammonium) propyl) indoleninium bromide (compound 2, m-3) (153 mg, 0.36 mmol), glutacone dialdehyde dianyl hydrochloride (compound 3) ( 114 mg, 0.4 mmol), acetic anhydride (2.2 ml) and acetic acid (1.6 ml) are heated for 2.5 hours at 105 ° C. At the end of heating, N- (5-carboxypentyl) -2,3,3-trimethyl-5-sulfoindoleninium (compound 4) (142 mg, 0.4 mmol), acetic anhydride (2.6 ml), acetic acid (1.3 ml) are added to the reaction mass. ), anhydrous potassium acetate (120 mg, 1.2 mmol) and heated for 4.5 h at 85 ° C. The solvent is removed, and the reaction product is isolated similarly to compound C1. 107 mg (39%) of compound D7 are obtained.

¹ H NMR (DMSO-d ₆ ) δ: 7.77 (m, 3H, δ-CH, β, β'-CH), 7.24, 7.39, 7.73 (3m, 6H, ArH), 6.53 (m, 2H, γ, γ'-CH), 6.36 (m, 2H, α, α'-CH), 4.05 (m, 4H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ , CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH ), 3.5 [m, 2H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ], 3.04 [s, 9H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ], 1.63 [2 s, 12H, CH ₃ (3.3), CH ₃ (3.3 ')], 2.05, 1.47, 1.31 (3m, 10H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ). Found: m / z 755.1 [M] ⁺ . C ₃₉ H ₅₁ N ₃ O ₈ S ₂ . Calculated: M = 753.97.

Example 19. Obtaining N- (7-aza-12-carboxy-6-oxododec-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (3-trimethylammoniopropyl) -5,5'- disulfoindotricarbocyanine dye (compound D8).

A mixture of N- (5-carboxypentyl) -3,3,3 ^, 3'-tetramethyl-N '- (3-trimethylammoniumpropyl) -5,5'-disulfoindotricarbocyanine dye (compound D7) (15 mg, 17.3 μmol) and bis β-nitrophenyl carbonate (13 mg, 43 μmol) was dissolved in DMSO (0.35 ml) and stirred for 1 h at ~ 20 ° C. Then the reaction mass is cooled to -18 ° C and a solution of 6-aminocaproic acid (34 mg, 0.26 mmol) in 0.1 M carbonate-bicarbonate buffer solution (0.35 ml) is introduced with stirring. The reaction mass is kept for 2 hours at ~ + 5 ° C, then diluted with 0.1 M TEAA. The target compound is isolated analogously to compound C1. 19 mg (84%) of compound D8 are obtained.

'H NMR (DMSO-d ₆ ) δ: 7.98 (m, 3H, δ-CH, β, β'-CH), 7.85, 7.61, 7.38 (3m, 6H, ArH), 6.56 (m, 2H, γ, γ'-CH), 6.35 (m, 2H, α, α'-CH), 4.08 (m, 4H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ , C (1) H ₂ ), 3.25 [m , 2H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ], 3.02 [s, 9H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ], 2.99 [m, 2H, C (8) H ₂ ] , 2.44 [m, 2H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ], 1.63 [s, 12H, CH ₃ (3.3), CH ₃ (3.3 ')], 2.02, 1.69, 1.49 , 1.32, 1.15 (5m, 16H, C (2) H ₂ , C (3) H ₂ , C (4) H ₂ , C (5) H ₂ , C (9) H ₂ , C (10) H ₂ , C (11) H ₂ , C (12) H ₂ ). Found: m / z 868.3 [M] ⁺ . C ₄₅ H ₆₂ N ₄ O ₉ S ₂ . Calculated: M = 867.13.

Example 20. Obtaining N- (7,14-diaza-19-carboxy-6,13-dioxononadec-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (3-trimethylammoniopropyl) -5 , 5'-disulfoindotricarbocyanine dye (compound D9).

A mixture of N-7-aza-12-carboxy-6-oxododecan-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (3-trimethylammoniumpropyl) -5,5'-disulfoindotricarbocyanine dye (compound D8) (15 mg, 17 μmol), bis-p-nitrophenyl carbonate (13 mg, 43 μmol) was dissolved in DMSO (0.35 ml) and stirred for 1 h at ~ 20 ° C. Then the reaction mass is cooled to -18 ° C and a solution of 6-aminocaproic acid (34 mg, 0.26 mmol) in 0.1 M carbonate-bicarbonate buffer solution (0.35 ml) is introduced with stirring. The reaction mass is kept for 2 hours at ~ + 5 ° C, then diluted with 0.1 M TEAA. The target compound is isolated analogously to compound C1. 15 mg (90%) of compound D9 are obtained.

'H NMR (DMSO-d ₆ ) δ: 8.05 (m, 3H, δ-CH, β, β'-CH), 7.81, 7.57, 7.41 (3m, 6H, ArH), 6.52 (m, 2H, γ, γ'-CH), 6.31 (m, 2H, α, α'-CH), 4.03 (m, 4H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ , C (1) H ₂ ), 3.55 [m , 2H, CH ₂ CH ₂ CH ₂ H (CH ₃ ) ₃ ], 3.08 [s, 9H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ], 2.91 [m, 4H, C (8) H ₂ , C (15) H ₂ ], 2.45 [m, 2H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ], 1.63 [s, 12H, CH ₃ (3.3), CH ₃ (3.3 ') ], 2.08, 1.87, 1.43, 1.33, 1.17 (5m, 24H, C (2) H ₂ , C (3) H ₂ , C (4) H ₂ , C (5) H ₂ , C (9) H ₂ , C (10) H ₂ , C (11) H ₂ , C (12) H ₂ , C (16) H ₂ , C (17) H ₂ , C (18) H ₂ , C (19) H ₂ ) . Found: m / z 981.3 [M] ⁺ . C ₅₁ H ₇₃ N ₅ O ₁₀ S ₂ . Calculated: M = 980.29.

Example 21. Obtaining N- (5-carboxypentyl) -3,3,3 ', 3'-tetramethyl-N' - (5-trimethylammoniopentyl) -5,5'-disulfoindotricarbocyanine dye (compound D10).

A mixture of 2,3,3-trimethyl-5-sulfo-N - ((5-trimethylammonium) pentyl) indoleninium bromide (compound 2, m = 5) (161 mg, 0.36 mmol), glutacone dialdehyde dianyl hydrochloride (compound 3) ( 114 mg, 0.4 mmol), acetic anhydride (2.2 ml) and acetic acid (1.6 ml) are heated for 2.5 hours at 105 ° C. At the end of heating, M- (5-carboxypentyl) -2,3,3-trimethyl-5-sulfoindoleninium (compound 4) (142 mg, 0.4 mmol), acetic anhydride (2.6 ml), acetic acid (1.3 ml) are added to the reaction mass. ), anhydrous potassium acetate (120 mg, 1.2 mmol) and heated for 4.5 h at 85 ° C. The solvent was removed and the reaction product was isolated similarly to compound C1. 121 mg (43%) of compound D10 are obtained.

'H NMR (DMSO-d ₆ ) δ: 7.87 (m, 3H, δ-CH, β, β'-CH), 7.29, 7.41, 7.71 (3m, 6H, ArH), 6.51 (m, 2H, γ, γ'-CH), 6.38 (m, 2H, α, α'-CH), 4.06 (m, 4H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ , CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH), 3.24 [m, 2H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ], 2.99 [s, 9H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ], 1.63 [s, 12H, CH ₃ (3.3), CH ₃ (3.3 ')], 2.02, 1.74, 1.49, 1.35 [4 m, 14H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ COOH, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ]. Found: m / z 783.2 [M] ⁺ . C ₄₁ H ₅₅ N ₃ O ₈ S ₂ . Calculated: M = 782.02.

Example 22. Obtaining N- (7-aza-12-carboxy-6-oxododec-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (3-trimethylammoniopentyl) -5,5'- disulfoindotricarbocyanine dye (compound D11).

A mixture of N- (5-carboxypentyl) -3,3,3 ', 3'-tetramethyl-N' - (5-trimethylammonium pentyl) -5,5'-disulfoindotricarbocyanine dye (compound D10) (12 mg, 15 μmol) and bis p-nitrophenyl carbonate (13 mg, 43 μmol) was dissolved in DMSO (0.35 ml) and stirred for 1 h at ~ 20 ° C. Then the reaction mass is cooled to -18 ° C and a solution of 6-aminocaproic acid (34 mg, 0.26 mmol) in 0.1 M carbonate-bicarbonate buffer solution (0.35 ml) is introduced with stirring. The reaction mass is kept for 2 hours at ~ + 5 ° C, then diluted with 0.1 M TEAA. The target compound is isolated analogously to compound C1. 19 mg (78%) of compound D11 are obtained.

¹ H NMR (DMSO-d6) δ: 8.03 (m, 3H, δ-CH, β, β'-CH), 7.82, 7.65, 7.41 (3m, 6H, ArH), 6.52 (m, 2H, γ, γ '-CH), 6.37 (m, 2H, α, α'-CH), 4.03 (m, 4H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₃ N (CH ₃ ) ₃ , C (1) H ₂ ), 3.26 [m, 2H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ], 2.97 [m, 4H, C (8) H ₂ , CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ], 1.66 [s, 12H, CH ₃ (3.3), CH ₃ (3.3 ')], 2.05, 1.66, 1.43, 1.36, 1.19 (5m, 22H, C (2) H ₂ , C (3) H ₂ , C (4) H ₂ , C (5) H ₂ , C (9) H ₂ , C (10) H ₂ , C (11) H ₂ , C (12) H ₂ , CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ). Found: m / z 896.3 [M] ⁺ . C ₄₇ H ₆₆ N ₄ O ₉ S ₂ . Calculated: M = 895.18.

Example 23. Obtaining N- (7,14-diaza-19-carboxy-6,13-dioxononadec-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (3-trimethylammoniopentyl) -5 , 5'-disulfoindotricarbocyanine dye (compound D12).

A mixture of N- (7-aza-12-carboxy-6-oxododecan-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (5-trimethylammoniumpental) -5,5'-disulfoindotricarbocyanine dye ( compound D11) (18 mg, 20 μmol), bis-p-nitrophenyl carbonate (13 mg, 43 μmol) was dissolved in DMSO (0.35 ml) and stirred for 1 h at ~ 20 ° C. Then the reaction mass is cooled to -18 ° C and a solution of 6-aminocaproic acid (34 mg, 0.26 mmol) in 0.1 M carbonate-bicarbonate buffer solution (0.35 ml) is introduced with stirring. The reaction mass is kept for 2 hours at ~ + 5 ° C, then diluted with 0.1 M TEAA. The target compound is isolated analogously to compound C1. 15 mg (78%) of compound D12 are obtained.

¹ H NMR (DMSO-d ₆ ) δ: 7.96 (m, 3H, δ-CH, β, β'-CH), 7.79, 7.52, 7.39 (3m, 6H, ArH), 6.51 (m, 2H, y, γ'-CH), 6.36 (m, 2H, α, α'-CH), 4.07 (m, 4H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ , C (1) H ₂ ), 3.22 [m , 2H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ ] N (CH ₃ ) ₃ ], 3.05 [s, 9H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ], 2.93 [m, 4H, C (8) H ₂ , C (15) H ₂ ], 2.48 [m, 2H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ], 1.65 [s, 12H, CH ₃ (3.3) , CH ₃ (3.3 ')], 2.05, 1.83, 1.47, 1.31, 1.23 (5m, 30H, C (2) H ₂ , C (3) H ₂ , C (4) H ₂ , C (5) H ₂ , C (9) H ₂ , C (10) H ₂ , C (11) H ₂ , C (12) H ₂ , C (16) H ₂ , C (17) H ₂ , C (18) H ₂ , C (19) H ₂ , CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ). Found: m / z 1009.5 [M] ⁺ . C ₅₃ H ₇₇ N ₅ O ₁₀ S ₂ . Calculated: M = 1008.34.

8.03 (m, 3H, δ-CH, β, β'-CH), 7.82, 7.65, 7.41 (3m, 6H, ArH), 6.52 (m, 2H, γ, γ'-CH), 6.37 (m, 2H , α, α'-CH),

Example 24. p-Nitrophenyl esters of dyes of General formula (B).

A mixture of dye (7.5 μmol), HBTU (8.5 mg, 22.5 μmol) and p-nitrophenol (5 mg, 37.5 μmol) was dissolved in DMF (0.6 ml) and stirred for 1.5 h at room temperature. Then the reaction mass was diluted with water (10 ml). The filtrate was purified by reverse phase chromatography (C18-RP) using an acetonitrile-water mixture as eluent. Received activated dye esters in quantitative yield (about 90%).

Example 25. Synthesis of fluorescently-labeled 5- (3-aminoallyl) -2′-deoxyuridine-5′-triphosphates of the general formula (A).

To a solution of the tetrasodium salt of 5- (3-aminoallyl) -2'-deoxyuridine-5'-triphosphate (AAdUTP) (1) (1 mg, 1.6 μmol) in NaHCO ₃ / Na ₂ CO ₃ buffer solution (0.2 ml, 0.1 M , pH 8.5) was added p-nitrophenyl ether dye of the general formula (B) (2 μmol) in DMF (0.2 ml) and the mixture was stirred for 12 hours at + 5 ° C. Conjugates of the general formula (A) were purified from excess dye (B) by precipitation with a 2% solution of NaClO ₄ in acetone (1.6 ml). The precipitate was collected by centrifugation at 13000 rpm for 1 min, washed with acetone (0.2 ml) and dissolved in 1 ml of a mixture of 30% acetonitrile in water.

A solution of the modified nucleotide of the general formula (A) in 1 ml of a mixture of 30% acetonitrile in water was applied to a column (2 × 8 cm) filled with DEAE cellulose (DE-52, Whatman) and eluted in a linear concentration gradient from 0.025M to 0.3 M TEANS (triethylammonium bicarbonate buffer solution) (pH 7.5) in 30% acetonitrile at a rate of 58 ml / hour. The target substance was collected from the column in a TEANS concentration range from 0.2 to 0.3 M.

Next, the sodium salt of the nucleotide of general formula (A) was obtained. For this, a fraction containing a modified nucleotide of the general formula (A) was collected, diluted to a volume of 50 ml with 0.1 M TEANS buffer solution (pH 8.5) and applied to a chromatographic column (2 × 8 cm) (Analtech, Newark, DE) filled with C18- RP (40-63 μm), washed with 0.1 M NaClO ₄ , 0.1 M EDTA (pH 8.0) and deionized water, and was eluted with a mixture of acetonitrile - water.

Modified nucleotides of the general formula (A) were prepared in different salt forms. For this, the procedure described above was performed, but at the stage of using 0.1 M NaClO ₄ , the following reagents were used:

- 0.1 M KCl - to obtain the potassium salt of the nucleotide;

- 0.1 M LiCl; - to obtain the lithium salt of the nucleotide;

- 0.1 M TEAA - to obtain the triethylammonium salt of the nucleotide;

- 0.1 M NH ₄ HCO ₃ - to obtain the ammonium salt of the nucleotide.

Fractions containing the target compound of general formula (A) were combined and diluted to a volume of 50 ml with deionized water. The concentration of the modified nucleotide of the general formula (A) was determined based on the molar extinction coefficient of cyanine dyes (B). Absorption was determined in triplicate by diluting the stock solution in MQ so that the optical density did not exceed 0.1.

The solvents were removed in vacuo, the resulting precipitate was dissolved in TE buffer (pH 8.0) to obtain a fluorescently labeled nucleotide solution of the general formula (A) with a concentration of 1 μmol / ml. The resulting solution was stored at a temperature of -20 ° C.

Example 26. Obtaining tetrasodium salt of 5- [4-aza-5-oxo-10- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3', 3 ' -tetramethyl-5,5'-disulfoindodicarbocyanin-1-yl) -dec-1-en-1-yl] -2'-deoxyuridine-5'-triphosphate (compound A1).

Obtain 1.2 μmol, yield 75% of compound A1. Found: m / z 1347.4 [M] ⁺ . C ₅₅ H ₇₄ N ₇ O ₂₂ P ₃ S ₂ ^4- . Calculated: M = 1342.26.

¹ H NMR (D ₂ O, 5, ppm, J / Hz): 1.35, 1.54, 1.79 (3 br.m., 14H, dye C (7) H ₂ , C (8) H ₂ , C (9) H ₂ , C (2 ') H ₂ , C (3') H ₂ , C (4 ') H ₂ , C (9') H ₂ ), 1.67 (s, dye 12H, C (3, 3) H ₃ , C (3 ', 3') H ₃ ), 2.24 (m, 6H, C (2 ') H ₂ , dye C (6) H ₂ , dye C (5') H ₂ ), 3.02 (s, 11H, N (CH ₃ ) ₃ , dye C (10 ') H ₂ ), 3.23 (m, 2H, dye C (8') H ₂ ), 3.81 (m, 2H, dye C (3) H ₂ ), 4.06 (br.m, 7H, C (3 ') H, C (5') H ₂ , dye C (10) H ₂ , dye C (1 ') H ₂ ), 4.57 (m, 1H, C (4 ') H), 6.26 (m, 5H, C (1') H, dye C (1) H, dye α, α'-CH, dye C (2) H), 6.59 (t, 1H, dye γ-CH, J 12.0 Hz), 7.33, 7.77 (2 m, 7Н, С (6) H, dye ArH), 8.05 (m, 2Н, dye β, β'-CH). NMR ³¹ P (D ₂ O, 6, ppm): -21.60 (t, ^β P), -10.76 (d, ^α P), -5.45 (d, ^γ P)

Example 27. Obtaining tetrasodium salt of 5- [4,11-diaz-5,12-dioxo-17- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3 ', 3'-tetramethyl-5,5'-disulfoindodicarbocyanin-1-yl) heptadec-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate (compound A2).

Obtain 1.02 μmol, yield 64% of compound A2. Found: m / z 1460.5 [M] ⁺ . C ₆₁ H ₈₅ N ₈ O ₂₃ P ₃ S ₂ ^4- . Calculated: M = 1455.42.

¹ H NMR (D ₂ O, 8, ppm, J / Hz): 1.27, 1.39, 1.56, 1.79 (4 br.m., 20H, dye C (7) H ₂ , C (8) H ₂ , C (9) H ₂ , C (14) H ₂ , C (15) H ₂ , C (16) H ₂ , dye C (2 ') H ₂ , C (3') H ₂ , C (4 ' ) H ₂ , C (9 ') H ₂ ), 1.67 (s, dye 12H, C (3,3) H ₃ , C (3', 3 ') H ₃ ), 2.19 (m, 6H, dye C ( 6) H ₂ , C (13) H ₂ , C (5 ') H ₂ ), 2.31 (m, 2H, C (2') H ₂ ), 3.04 (s, 11H, NCH ₃ ) ₃ dye C (10 ') H ₂ ), 3.22 (m, 4H, dye C (10) H ₂ , dye C (8') H ₂ ,), 3.82 (m, 2H, dye C (3) H ₂ ), 4.06 (br. m, 8H, C (3 ') H, C (4') H, C (5 ') H ₂ , dye C (1') H ₂ , dye C (17) H ₂ ), 6.25 (m, 5H, C (1 ') H, dye α, α'-CH, dye C (1) H, C (2) H), 6.58 (m, 1H, dye γ-CH), 7.30, 7.84 (2 m, 7H , C (6) H, dye ArH), 8.06 (m, 2H, dye β, β'-CH). NMR ³¹ P (D ₂ O, 8, ppm): -21.54 (t, ^β P), -10.79 (d, ^α P), -5.53 (d, ^γ P)

Example 28. Obtaining the tetrasodium salt of 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3 , 3,3 ', 3'-tetramethyl-5,5'-disulfoindodicarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate (compound A3).

Obtain 1.25 μmol, yield 78% of compound A3. Found: m / z 1573.8 [M] ⁺ . C ₆₇ H ₉₆ N ₉ O ₂₄ P ₃ S ₂ ^4- . Calculated: M = 1568.58.

¹ H NMR (D ₂ O, 5, ppm, J / Hz): 1.23, 1.38, 1.54, 1.78 (4 brpm, 26H, dye C (7) H ₂ , C (8) H ₂ , C (9) H ₂ , C (14) H ₂ , C (15) H ₂ , C (16) H ₂ , C (21) H ₂ , C (22) H ₂ , C (23) H ₂ , C (2 ') H ₂ , C (3') H ₂ , C (4 ') H ₂ , C (9') H ₂ ), 1.65 (s, dye 12H, C (3.3) H ₃ , C (3 ', 3') H ₃ ), 2.22 (m, 8H, dye C (6) H ₂ , C (13) H ₂ , C (20) H ₂ , dye C (5 ') H ₂ ), 2.34 (m, 2H, C (2 ') H ₂ ), 3.08 (s, 11H, N (CH ₃ ) ₃ , dye C (10') H ₂ ), 3.22 (m, 6H, dye C (10) H ₂ , C (17) H ₂ , dye C (8 ') H ₂ ), 3.84 (m, 2H, dye C (3) H ₂ ), 4.09 (br. M, 8H, C (3') H, C ( 4 ') H, C (5') H ₂ , dye C (1 ') H ₂ , dye C (24) H ₂ ), 6.27 (m, 5H, C (1') H, dye α, α'- CH, dye C (1) H, C (2) H), 6.56 (m, 1H, dye γ-CH), 7.30, 7.88 (2 m, 7H, C (6) H, dye ArH), 8.09 ( m, 2H, dye β, β'-CH). NMR ³¹ P (D ₂ O, δ, ppm): -21.51 (t, ^β P), -10.82 (d, ^α P), -5.59 (d, ^γ P)

Example 29. Obtaining tetrasodium salt of 5- [4-aza-5-oxo-10- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindodicarbocyanine 1-yl) -dec-1-en-1-yl] -2'-deoxyuridin-5'-triphosphate (compound A4).

Obtain 1.3 μmol (81%) of compound A4. Found: m / z 1234.3 [M] ⁺ . C ₄₉ H ₆₃ N ₆ O ₂₁ P ₃ S ₂ ^4- . Calculated: M = 1229.11.

¹ H NMR (D ₂ O, 5, ppm, J / Hz): 1.32, 1.57, 1.75 (3 m, 6H, dye C (7) H ₂ , C (8) H ₂ , C (9) H ₂ ), 1.63 (s, dye 12H, C (3.3) H ₃ , C (3 ', 3') H ₃ ), 2.29 (m, 6H, C (2 ') H ₂ , dye C (6 ) H ₂ , CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.05 (s, 9H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.48 (s, 2H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.83 (m, 2H, dye C (3) H ₂ ), 3.91-4.28 (br.m, 8H, C (3 ') H, C (4') H, C (5 ') H ₂ , CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ , dye C (10) H ₂ ), 6.12 (m, 3H, dye α, α'-CH, C (1') H), 6.31 (m, 2H, dye C (1) H, C (2) H), 6.47 (m, 1H, dye γ-CH), 7.11, 7.23, 7.39, 7.68, 7.74 (5m, 7H, C (6) H, ArH), 7.99 (m, 2H, dye β, β'-CH). NMR ³¹ P (D ₂ O, 8, ppm): -22.94 (t, ^β P), - 11.38 (d, ^α P), -8.94 (d, ^γ P)

Example 30. Obtaining tetrasodium salt of 5- [4,11-diaza-5,12-dioxo-17- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl-5.5 '-disulfoindodicarbocyanin-1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridin-5'-triphosphate (compound A5).

Obtain 1.1 μmol (69%) of compound A5. Found: m / z 1347.4 [M] ⁺ . C ₅₅ H ₇₄ N ₇ O ₂₂ P ₃ S ₂ ^4- . Calculated: M = 1342.26.

¹ H NMR (D ₂ O, 5, ppm, J / Hz): 1.29, 1.52, 1.74 (3 br.m., 12H, dye C (7) H ₂ , C (8) H ₂ , C (9) H ₂ , C (14) H ₂ , C (15) H ₂ , C (16) H ₂ ), 1.65 (s, dye 12H, C (3,3) H ₃ , C (3 ', 3 ') H ₃ ), 2.29 (m, 8H, C (2') H ₂ , dye C (6) H ₂ , C (13) H ₂ , CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 2.99 (m, 2H, dye C (10) H ₂ ), 3.08 (s, 9H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.48 (s, 2H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.83 (m, 2H, dye C (3) H ₂ ), 4.08 (br.m, 8H, C (3 ') H, C (4') H, C (5 ') H ₂ , CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ , dye C (17) H ₂ ), 6.16 (m, 3H, dye α, α'-CH, C (1 ') H), 6.34 (m, 2H, dye C (1) H, C (2) H), 6.49 (m, 1H, dye γ-CH), 7.19, 7.25, 7.44, 7.68, 7.79 (5m, 7H, C (6) H, ArH), 8.05 (m, 2H, dye β, β'-CH). NMR ³¹ P (D ₂ O, 5, ppm): -21.64 (t, ^β P), -10.76 (d, ^α P), -7.64 (d, ^γ P)

Example 31. Obtaining the tetrasodium salt of 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl -5,5'-disulfoindodicarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridin-5'-triphosphate (compound A6).

Obtain 1 μmol (63%) of compound A6. Found: m / z 1460.5 [M] ⁺ . C ₆₁ H ₈₅ N ₈ O ₂₃ P ₃ S ₂ ^4- . Calculated: M = 1455.42.

¹ H NMR (D ₂ O, δ, ppm, J / Hz): 1.21, 1.48, 1.76 (3 br.m., 18H, dye C (7) H ₂ , C (8) H ₂ , C (9) H ₂ , C (14) H ₂ , C (15) H ₂ , C (16) H ₂ , C (21) H ₂ , C (22) H ₂ , C (23) H ₂ ), 1.66 (s, dye 12H, C (3.3) H ₃ , C (3 ', 3') H ₃ ), 2.29 (m, 10H, C (2 ') H ₂ , dye C (6) H ₂ , C (13) H ₂ , C (20) H ₂ , CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.01 (m, 4H, dye C (10) H ₂ , C (17) H ₂ ), 3.12 (s, 9H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.51 (s, 2H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.83 (m, 2H, dye C (3 ) H ₂ ), 4.08 (br. M, 8H, C (3 ') H, C (4') H, C (5 ') H ₂ , CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ , dye C (24) H ₂ ), 6.19 (m, 3H, dye α, α'-C H , C (1 ') H), 6.37 (m, 2H, dye C (1) H, C (2) H), 6.51 (m, 1H, dye γ-CH), 7.21, 7.28, 7.39, 7.62, 7.74 (5m, 7H, C (6) H, ArH), 8.01 (m, 2H, dye β, β'-CH) . NMR ³¹ P (D ₂ O, 8, ppm): -21.14 (t, ^β P), -10.05 (d, ^α P), -7.86 (d, ^γ P).

Example 32. Obtaining the tetrasodium salt of 5- [4-aza-5-oxo-10- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindodicarbocyanine - 1-yl) - dec-1-en-1-yl] -2'-deoxyuridin-5'-triphosphate (compound A7).

Obtain 1.2 μmol (76%) of compound A7. Found: m / z 1258.4 [M] ⁺ . C ₅₁ H ₆₇ N ₆ O ₂₁ P ₃ S ₂ ^4- . Calculated: M = 1257.16.

¹ H NMR (D ₂ O, 5, ppm, J / Hz): 1.29, 1.53, 1.72 (3 br.m., 10H, dye C (7) H ₂ , C (8) H ₂ , C (9) H ₂ , CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 1.61 (s, dye 12H, C (3.3) H ₃ , C (3 ', 3') H ₃ ), 2.29 (m, 4H, C (2 ') H ₂ , dye C (6) H ₂ ), 3.01 (s, 9H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.25 (s, 2H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.79 (m, 2H, dye C (3) H ₂ ), 4.13 (br m, 8H, C (3 ') ) H, C (4 ') H, C (5') H ₂ , CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ , dye C (10) H ₂ ), 6.15 (m, 3H, dye α, α'-CH, C (1 ') H), 6.31 (m, 2H, dye C (1) H, C (2) H), 6.49 (m, 1H, dye γ-CH), 7.15, 7.23, 7.34, 7.63, 7.71 (5m, 7H, C (6) H, ArH), 7.95 (m, 2H, dye β, β'-CH). NMR ³¹ P (D ₂ O, δ, ppm): -20.84 (t, ^β P), -10.13 (d, ^α P), -7.03 (d, ^γ P).

Example 33. Obtaining the tetrasodium salt of 5- [4,11-diaza-5,12-dioxo-17- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5.5 '-disulfoindodicarbocyanin-1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridin-5'-triphosphate (compound A8).

Obtain 1 μmol (64%) of compound A8. Found: m / z 1375.4 [M] ⁺ . C ₅₇ H ₇₈ N ₇ O ₂₂ P ₃ S ₂ ^4- . Calculated: M = 1370.32.

¹ H NMR (D ₂ O, δ, ppm, J / Hz): 1.24, 1.51, 1.69 (3 br.m., 14H, dye C (7) H ₂ , C (8) H ₂ , C (9) H ₂ , C (14) H ₂ , C (15) H ₂ , C (16) H ₂ , CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 1.64 (s, dye 12H, C (3.3) H ₃ , C (3 ', 3') H ₃ ), 2.23 (m, 6H, C (2 ') H ₂ , dye C (6) H ₂ , C (13) H ₂ ), 2.99 (m, 2H, dye C (10) H ₂ ), 3.11 (s, 9H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.25 (s, 2H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.75 (m, 2H, dye C (3) H ₂ ), 4.12 (br.m, 8H, C (3 ') H, C (4 ') H, C (5') H ₂ , CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ , dye C (17) H ₂ ), 6.15 (m, 3H, dye α, α'- CH, C (1 ') H), 6.33 (m, 2H, dye C (1) H, C (2) H), 6.51 (m, 1H, dye γ-CH), 7.13, 7.21, 7.32, 7.61, 7.69 (5m, 7H, C (6) H, ArH); 7.98 (m, 2H, dye β, β'-CH). NMR ³¹ P (D ₂ O, δ, ppm): -20.34 (t, ^β P), -10.89 (d, ^α P), -7.73 (d, ^γ P).

Example 34. Obtaining the tetrasodium salt of 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl -5,5'-disulfoindodicarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridin-5'-triphosphate (compound A9).

Obtain 1.3 μmol (82%) of compound A9. Found: m / z 1488.6 [M] ⁺ . C ₆₃ H ₈₉ N ₈ O ₂₃ P ₃ S ₂ ^4- . Calculated: M = 1483.47.

¹ H NMR (D ₂ O, 5, ppm, J / Hz): 1.21, 1.48, 1.63 (3 br.m., 20H, dye C (7) H ₂ , C (8) H ₂ , C (9) H ₂ , C (14) H ₂ , C (15) H ₂ , C (16) H ₂ , C (21) H ₂ , C (22) H ₂ , C (23) H ₂ , CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 1.59 (s, dye 12H, C (3.3) H ₃ , C (3 ', 3') H ₃ ), 2.21 (m, 6H, C (2 ') H ₂ , dye C (6) H ₂ , C (13) H ₂ , C (20) H ₂ ), 2.99 (m, 4H, dye C (10) H ₂ , C (17) H ₂ ), 3.08 (s, 9H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.27 (s, 2H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.71 (m, 2H, dye C (3) H ₂ ), 4.08 (br.m, 8H, C (3 ') H, C (4') H, C (5 ') H ₂ , CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ , dye C (24) H ₂ ), 6.11 (m, 3H, dye α, α'-CH, C (1 ') H), 6.35 (m, 2H , dye C (1) H, C (2) H), 6.54 (m, 1H, dye γ-CH), 7.11, 7.25, 7.36, 7.59, 7.74 (5m, 7H, C (6) H, ArH) 8.08 (m, 2H, dye β, β'-CH). NMR ³¹ P (D ₂ O, δ, ppm): -20.86 (t, ^β P), - 11.63 (d, ^α P), -7.08 (d, ^γ P).

Example 35. Obtaining tetrasodium salt of 5- [4-aza-5-oxo-10- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3', 3 ' -tetramethyl-5,5'-disulfoindotricarbocyanin-1-yl) -dec-1-en-1-yl] -2'-deoxyuridine-5'-triphosphate (compound A10).

0.93 μmol is obtained, yield 58% of compound A10. Found: m / z 1373.4 [M] ⁺ . C ₅₇ H ₇₆ N ₇ O ₂₂ P ₃ S ₂ ^4- . Calculated: M = 1368.3.

¹ H NMR (D ₂ O, δ, ppm, J / Hz): 1.35, 1.54, 1.68, 1.79, 2.03 (5 br.m, 12H, dye C (7) H ₂ , C (8) H ₂ , C (9) H ₂ , C (2 ') H ₂ , C (3') H ₂ , C (4 ') H ₂ ), 1.64 (s, dye 12H, C (3,3) H ₃ , C (3 ', 3') H ₃ ), 2.24 (m, 6H, C (2 ') H ₂ , dye C (6) H ₂ , dye C (5') H ₂ ), 3.02 (s, 11H , N (CH ₃ ) ₃ , dye C (10 ') H ₂ ), 3.23 (m, 4H, dye C (8') H ₂ , C (9 ') H ₂ ), 3.81 (m, 2H, dye C (3) H ₂ ), 4.06 (br.m, 7H, C (3 ') H, C (5') H ₂ , dye C (10) H ₂ , dye C (1 ') H ₂ ), 4.57 ( m, 1H, C (4 ') H), 6.32 (m, 5H, C (1') H, dye C (1) H, dye α, α'-CH, dye C (2) H), 6.53 ( m, 2H, dye γ, γ'-CH), 7.31, 7.48, 7.79 (3m, 7H, C (6) H, dye ArH), 7.89 (m, 4H, dye β, β'-CH, dye δ- CH). NMR ³¹ P (D ₂ O, 6, ppm): -21.85 (t, ^β P), -10.54 (d, ^α P), -5.32 (d, ^γ P)

Example 36. Obtaining tetrasodium salt of 5- [4,11-diaza-5,12-dioxo-17- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3 ', 3'-tetramethyl-5,5'-disulfoindotricarbocyanin-1-yl) heptadec-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate (compound A11).

1.17 μmol is obtained, yield 73% of compound A11. Found: m / z 146.6 [M] ⁺ . C ₆₃ H ₈₇ N ₈ O ₂₃ P ₃ S ₂ ^4- . Calculated: M = 1481.46.

¹ H NMR (D ₂ O, 5, ppm, J / Hz): 1.29, 1.52, 1.75 (3 br.m., 18H, dye C (7) H ₂ , C (8) H ₂ , C (9) H ₂ , C (14) H ₂ , C (15) H ₂ , C (16) H ₂ , dye C (2 ') H ₂ , C (3') H ₂ , C (4 ') H ₂ ), 1.62 (s, dye 12H, C (3.3) H ₃ , C (3 ', 3') H ₃ ), 2.17 (m, 6H, dye C (6) H ₂ , C (13) H ₂ , C (5 ') H ₂ ), 2.31 (m, 2H, C (2') H ₂ ), 3.01 (s, 11H, N (CH) ₃ dye C (10 ') H ₂ ), 3.25 (m , 6H, dye C (10) H ₂ , dye C (8 ') H ₂ , dye C (9') H ₂ ), 3.81 (m, 2H, dye C (3) H ₂ ), 4.02 (br. M , 8H, C (3 ') H, C (4') H, C (5 ') H ₂ , dye C (1') H ₂ , dye C (17) H ₂ ), 6.27 (m, 5H, C (1 ') H, dye α, α'-CH, dye C (1) H, C (2) H), 6.55 (m, 2H, dye γ, γ'-CH), 7.36, 7.84 (2 br. m., 7H, C (6) H, dye ArH), 7.82 (m, 3H, dye δ-CH, β, β'-CH). NMR ³¹ P (D ₂ O, 5, ppm): -21.39 (t, ^β P), -10.46 (d, ^α P), -5.14 (d, ^γ P).

Example 37. Obtaining the tetrasodium salt of 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3 , 3,3 ', 3'-tetramethyl-5,5'-disulfoindotricarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate (compound A12).

Obtain 0.98 μmol, yield 61% of compound A12. Found: m / z 1599.8 [M] ⁺ . C ₆₉ H ₉₈ N ₉ O ₂₄ P ₃ S ₂ ^4- . Calculated: M = 1594.62.

¹ H NMR (D ₂ O, δ, ppm, J / Hz): 1.28, 1.34, 1.51, 1.76 (4 br.m., 26H, dye C (7) H ₂ , C (8) H ₂ , C (9) H ₂ , C (14) H ₂ , C (15) H ₂ , C (16) H ₂ , C (21) H ₂ , C (22) H ₂ , C (23) H ₂ , C (2 ') H ₂ , C (3') H ₂ , C (4 ') H ₂ , C (9') H ₂ ), 1.62 (s, dye 12H, C (3,3) H ₃ , C (3 ', 3') H ₃ ), 2.16 (m, 8H, dye C (6) H ₂ , C (13) H ₂ , C (20) H ₂ , dye C (5 ') H ₂ ), 2.31 (m, 2H, C (2 ') H ₂ ), 3.12 (s, 11H, N (CH ₃ ) ₃ , dye C (10') H ₂ ), 3.25 (m, 6H, dye C (10) H ₂ , C (17) H ₂ , dye C (8 ') H ₂ ), 3.82 (m, 2H, dye C (3) H ₂ ), 4.03 (br.m, 8H, C (3') H, C ( 4 ') H, C (5') H ₂ , dye C (1 ') H ₂ , dye C (24) H ₂ ), 6.32 (m, 5H, C (1') H, dye α, α'- CH, dye C (1) H, C (2) H), 6.52 (m, 2H, dye γ, γ'-CH), 7.25, 7.64 (2 br.m., 7H, C (6) H, dye ArH), 8.09 (m, 3H, dye δ-CH, β, β'-CH). NMR ³¹ P (D ₂ O, δ, ppm): -21.13 (t, ^β P), -10.64 (d, ^α P), -5.53 (d, ^γ P)

Example 38. Obtaining tetrasodium salt of 5- [4-aza-5-oxo-10- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindotricarbocyanine - 1-yl) -dec-1-en-1-yl] -2'-deoxyuridin-5'-triphosphate (compound A13).

1.18 μmol (74%) of compound A13 is obtained. Found: m / z 1260.2 [M] ⁺ . C ₅₁ H ₆₅ N ₆ O ₂₁ P ₃ S ₂ ^4- . Calculated: M = 1255.14.

¹ H NMR (D ₂ O, 5, ppm, J / Hz): 1.38, 1.54, 1.71 (3 br.m, 6H, dye C (7) H ₂ , C (8) H ₂ , C ( 9) H ₂ ), 1.66 (s, dye 12H, C (3.3) H ₃ , C (3 ', 3') H ₃ ), 2.15 (m, 6H, C (2 ') H ₂ , dye C (6) H ₂ , CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.08 (s, 9H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.42 (s, 2H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.81 (m, 2H, dye C (3) H ₂ ), 4.15 (br.m, 8H, C (3 ') H, C (4') H, C (5 ') H ₂ , CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ , dye C (10) H ₂ ), 6.23 (m, 3H, dye α, α'-CH, C (1') H), 6.31 (m, 2H, dye C (1) H, C (2) H), 6.55 (m, 2H, dye γ, γ'-CH), 7.28, 7.32, 7.61, 7.77 (5m, 7H, C (6) H, ArH), 7.99 (m, 3H, dye δ-CH, β, β'-CH). NMR ³¹ P (D ₂ O, 5, ppm): -22.14 (t, ^β P), -11.57 (d, ^α P), -8.73 (d, ^γ P).

Example 39. Obtaining the tetrasodium salt of 5- [4,11-diaza-5,12-dioxo-17- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl-5.5 '-disulfoindotricarbocyanin-1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridin-5'-triphosphate (compound A14).

Obtain 0.98 μmol (61%) of compound A14. Found: m / z 1373.4 [M] ⁺ . C ₅₇ H ₇₆ N ₇ O ₂₂ P ₃ S ₂ ^4- . Calculated: M = 1368.3.

¹ H NMR (D ₂ O, δ, ppm, J / Hz): 1.24, 1.58, 1.71 (3 br.m., 12H, dye C (7) H ₂ , C (8) H ₂ , C (9) H ₂ , C (14) H ₂ , C (15) H ₂ , C (16) H ₂ ), 1.58 (s, dye 12H, C (3.3) H ₃ , C (3 ', 3 ') H ₃ ), 2.25 (m, 8H, C (2') H ₂ , dye C (6) H ₂ , C (13) H ₂ , CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.05 (m, 2H, dye C (10) H ₂ ), 3.13 (s, 9H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.42 (s, 2H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.87 (m, 2H, dye C (3) H ₂ ), 4.13 (br.m, 8H, C (3 ') H, C (4') H, C (5 ') H ₂ , CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ , dye C (17) H ₂ ), 6.25 (m, 3H, dye α, α'-CH, C (1 ') H), 6.46 (m, 5H, dye C (1) H, C (2) H), dye γ, γ'-CH), 7.25, 7.41, 7.62, 7.71 (4m, 7H, C (6) H, ArH), 8.05 (m, 3H, dye δ-CH, β, β'-CH). NMR ³¹ P (D ₂ O, δ, ppm): -21.66 (t, ^β P), -10.62 (d, ^α P), -7.53 (d, ^γ P).

Example 40. Obtaining the tetrasodium salt of 5- [4,11,18-triaz-5,12,19-trioxo-24- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl -5,5'-disulfoindotricarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridin-5'-triphosphate (compound A15).

Obtain 1.1 μmol (69%) of compound A15. Found: m / z 1486.6 [M] ⁺ . C ₆₃ H ₈₇ N ₈ O ₂₃ P ₃ S ₂ ^4- . Calculated: M = 1481.46.

¹ H NMR (D ₂ O, 6, ppm, J / Hz): 1.23, 1.42, 1.79 (3 rpm, 18H, dye C (7) H ₂ , C (8) H ₂ , C (9) H ₂ , C (14) H ₂ , C (15) H ₂ , C (16) H ₂ , C (21) H ₂ , C (22) H ₂ , C (23) H ₂ ), 1.63 (s, dye 12H, C (3.3) H ₃ , C (3 ', 3') H ₃ ), 2.23 (m, 10H, C (2 ') H ₂ , dye C (6) H ₂ , C (13) H ₂ , C (20) H ₂ , CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.07 (m, 4H, dye C (10) H ₂ , C (17) H ₂ ), 3.18 (s, 9H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.49 (s, 2H, CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.81 (m, 2H, dye C (3 ) H ₂ ), 4.02 (br. M, 8H, C (3 ') H, C (4') H, C (5 ') H ₂ , CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ , dye C (24) H ₂ ), 6.26 (m, 3H, dye α, α'-C H , C (1 ') H), 6.48 (m, 4H, dye C (1) H, C (2) H, dye γ, γ'-CH), 7.23, 7.31, 7.54 (3m, 7H, C (6) H, ArH), 7.99 (m, 3H, dye δ-CH, β, β'-C). NMR ³¹ P (D ₂ O, 5, ppm): -21.68 (t, ^β P), -10.32 (d, ^α P), -7.45 (d, ^γ P).

Example 41. Obtaining the tetrasodium salt of 5- [4-aza-5-oxo-10- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindotricarbocyanine - 1-yl) - dec-1-en-1-yl] -2'-deoxyuridin-5'-triphosphate (compound A16).

1.15 μmol (72%) of compound A16 is obtained. Found: m / z 1288.3 [M] ⁺ . C ₅₃ H ₆₉ N ₆ O21P ₃ S ₂ ^4- . Calculated: M = 1283.20.

¹ H NMR (D ₂ O, 5, ppm, J / Hz): 1.25, 1.46, 1.72 (3 br.m., 10H, dye C (7) H ₂ , C (8) H ₂ , C (9) H ₂ , CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 1.65 (s, dye 12H, C (3.3) H ₃ , C (3 ', 3') H ₃ ), 2.25 (m, 4H, C (2 ') H ₂ , dye C (6) H ₂ ), 3.06 (s, 9H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.22 (s, 2H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.71 (m, 2H, dye C (3) H ₂ ), 4.09 (br m, 8H, C (3 ') ) H, C (4 ') H, C (5') H ₂ , CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ , dye C (10) H ₂ ), 6.31 (m, 3H, dye α, α'-CH, C (1 ') H), 6.49 (m, 4H, dye C (1) H, C (2) H, dye γ, γ'-CH), 7.21, 7.32, 7.68 ( 4 m, 7H, C (6) H, ArH), 7.95 (m, 3H, dye δ-CH, β, β'-CH). NMR ³¹ P (D ₂ O, δ, ppm): -21.34 (t, ^β P), -10.27 (d, ° P), -7.93 (d ^γ P).

Example 42. Obtaining the tetrasodium salt of 5- [4,11-diaz-5,12-dioxo-17- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5.5 '-disulfoindotricarbocyanin-1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridin-5'-triphosphate (compound A17).

Obtain 1.09 μmol (68%) of compound A17. Found: m / z 1401.5 [M] ⁺ . C ₅₉ H ₈₀ N ₇ O ₂₂ P ₃ S ₂ ^4- . Calculated: M = 1396.35.

¹ H NMR (D ₂ O, δ ppm, J / Hz): 1.28, 1.41, 1.56 (3 br.m., 14H, dye C (7) H ₂ , C (8) H ₂ , C ( 9) H ₂ , C (14) H ₂ , C (15) H ₂ , C (16) H ₂ , CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 1.68 (s, dye 12H , C (3.3) H ₃ , C (3 ', 3') H ₃ ), 2.27 (m, 6H, C (2 ') H ₂ , dye C (6) H ₂ , C (13) H ₂ ), 2.84 (m, 2H, dye C (10) H ₂ ), 3.05 (s, 9H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.18 (s, 2H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.72 (m, 2H, dye C (3) H ₂ ), 4.03 (br m, 8H, C (3 ') H, C (4' ) H, C (5 ') H ₂ , CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ , dye C (17) H ₂ ), 6.32 (m, 3H, dye α, α'-CH , C (1 ') H), 6.46 (m, 4H, dye C (1) H, C (2) H, dye γ, γ'-CH), 7.25, 7.31, 7.65 (3m, 7H, C ( 6) H, ArH), 7.98 (m, 3H, dye δ-CH, β, β'-CH). NMR ³¹ P (D ₂ O, 6, ppm): -20.94 (t, ^β P), - 10.12 (d, ^α P), -7.64 (d, ^β P).

Example 43. Obtaining tetrasodium salt of 5- [4,11,18-triaz-5,12,19-trioxo-24- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl -5,5'-disulfoindotricarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate (compound A18).

Obtain 1.2 μmol (75%) of compound A18. Found: m / z 1514.7 [M] ⁺ . C ₆₅ H ₉₁ N ₈ O ₂₃ P ₃ S ₂ ^4- . Calculated: M = 1509.51.

¹ H NMR (D ₂ O, δ, ppm, J / Hz): 1.27, 1.42, 1.61 (3 br.m., 20H, dye C (7) H ₂ , C (8) H ₂ , C (9) H ₂ , C (14) H ₂ , C (15) H ₂ , C (16) H ₂ , C (21) H ₂ , C (22) H ₂ , C (23) H ₂ , CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 1.57 (s, dye 12H, C (3.3) H ₃ , C (3 ', 3') H ₃ ), 2.18 (m, 6H, C (2 ') H ₂ , dye C (6) H ₂ , C (13) H ₂ , C (20) H ₂ ), 2.95 (m, 4H, dye C (10) H ₂ , C (17) H ₂ ), 3.12 (s, 9H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.21 (s, 2H, CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ ), 3.69 (m, 2H, dye C (3) H ₂ ), 4.02 (br.m, 8H, C (3 ') H, C (4') H, C (5 ') H ₂ , CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ N (CH ₃ ) ₃ , dye C (24) H ₂ ), 6.32 (m, 3H, dye α, α'-CH, C (1 ') H), 6.46 (m, 2H dye C (1) H, C (2) H, dye γ, γ'-CH), 7.29, 7.38, 7.64 (3 m, 7Н, С (6) H, ArH), 8.02 (m, 3Н, dye δ-CH, β, β'-CH). NMR ³¹ P (D ₂ O, δ, ppm): -21.15 (t, ^β P), -10.94 (d, ^α P), -7.86 (d, ^γ P).

Example 44. The use of fluorescently-labeled nucleoside triphosphates of the general formula (A) using the KRAS-biochip test system as an example (Biochip-IMB, Moscow, Russia).

DNA samples were analyzed in accordance with the manufacturer's recommendations (Biochip-IMB, Moscow, Russia) (M. A. Emelyanova. Determination of mutations in the KRAS gene in tumor cells using biological microarrays // Molecular Biology. 2011. - V. 45, No. 5. - S. 863-870). The detection of PCR products after each step was confirmed by agarose gel electrophoresis.

At the first stage of multiplex PCR, genomic DNA was amplified in a final volume of 25 μl. The reaction mixture (final volume 25 μl) contained 3 μl Taq buffer, 1.5 mm MgCl ₂ , 2.5 units Taq polymerase, 0.2 μM of each triphosphate deoxynucleoside, 0.2 μM primers, 0.03 μM LNA oligonucleotide and 25 ng DNA. The temperature regime was as follows: denaturation 95 ° С - 3.5 min, then 35 amplification cycles (94 ° С - 30 sec, 60 ° С - 20 sec, 72 ° С - 10 sec), the final stage of incubation 72 ° С - 3 min. 2.5 μl of the reaction mixture obtained in stage 1 was used as a matrix for carrying out 2 stages. The second stage was carried out by the method of asymmetric multiplex PCR to obtain the predominant fluorescently-labeled product necessary for hybridization with oligonucleotide probes. The reaction mixture of the 2nd stage contained 0.2 nmol of a fluorescently labeled nucleotide of the general formula (A) and an excess of one primer. The temperature regime was as follows: denaturation 95 ° С - 3.5 min, then 35 amplification cycles (94 ° С - 30 sec, 60 ° С - 20 sec, 72 ° С - 10 sec), the final stage of incubation 72 ° С - 3 min. To 12 μl of the reaction mixture obtained in stage 2 of multiplex PCR and containing mainly single-stranded fluorescently-labeled DNA, 24 μl of a concentrated solution of hybridization buffer was added. The resulting mixture was placed in a hybridization chamber of the biochip. Hybridization was carried out at 37 ° C for 18 hours. At the end of hybridization, the microchip was washed with distilled water and dried in air. The hybridization results were recorded on a Chipdetector portable fluorescence analyzer (Biochip-IMB, Moscow, Russia) using specialized Imageware software.

Claims (137)

1. The compound of General formula

, where Y represents - (CH ₂ ) ₃ -; - (CH ₂ ) ₅ -; - (CH ₂ ) ₅ CONH (CH ₂ ) ₃ -; X represents —CH ₂ -; -CH ₂ CONH (CH ₂ ) ₅ -; -CH ₂ CONH (CH ₂ ) ₅ CONH (CH ₂ ) ₅ -; M ⁺ represents H ⁺ , Na ⁺ , Li ⁺ , K ⁺ , [CH ₃ (CH ₂ ) _n ] _(4-m) NH _m ⁺ (where n = 0-5, m = 0-4). 2. The compounds of claim 1, which are tetrasodium salt of 5- [4-aza-5-oxo-10- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3', 3'-tetramethyl-5 , 5'-disulfoindodicarbocyanin-1-yl) -dec-1-en-1-yl] -2'-deoxyuridine-5'-triphosphate, 5- [4-aza-5-oxo-10- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3', 3'-tetramethyl-tetra potassium salt , 5'-disulfoindodicarbocyanin-1-yl) -dec-1-en-1-yl] -2'-deoxyuridine-5'-triphosphate, tetralithium salt of 5- [4-aza-5-oxo-10- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3', 3'-tetramethyl-5 , 5'-disulfoindodicarbocyanin-1-yl) -dec-1-en-1-yl] -2'-deoxyuridine-5'-triphosphate, 5- [4-aza-5-oxo-10- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3', 3'-tetramethyl-5 tetraammonium salt , 5'-disulfoindodicarbocyanin-1-yl) -dec-1-en-1-yl] -2'-deoxyuridine-5'-triphosphate, tetrakis (triethylammonium) salt 5- [4-aza-5-oxo-10- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3', 3'- tetramethyl-5,5'-disulfoindodicarbocyanin-1-yl) -dec-1-en-1-yl] -2'-deoxyuridine-5'-triphosphate, tetrasodium salt of 5- [4,11-diaza-5,12-dioxo-17- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3', 3 ' -tetramethyl-5,5'-disulfoindodicarbocyanin-1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, 5- [4,11-diaza-5,12-dioxo-17- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3' tetra potassium salt -tetramethyl-5,5'-disulfoindodicarbocyanin-1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetralithium salt of 5- [4,11-diaza-5,12-dioxo-17- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3', 3 ' -tetramethyl-5,5'-disulfoindodicarbocyanin-1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetraammonium salt of 5- [4,11-diaza-5,12-dioxo-17- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3', 3 ' -tetramethyl-5,5'-disulfoindodicarbocyanin-1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetrakis (triethylammonium) salt 5- [4,11-diaza-5,12-dioxo-17- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3' , 3'-tetramethyl-5,5'-disulfoindodicarbocyanin-1-yl) heptadec-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetrasodium salt of 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3 ', 3'-tetramethyl-5,5'-disulfoindodicarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3 tetra potassium salt ', 3'-tetramethyl-5,5'-disulfoindodicarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetralithium salt of 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3 ', 3'-tetramethyl-5,5'-disulfoindodicarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3 tetraammonium salt ', 3'-tetramethyl-5,5'-disulfoindodicarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetrakis (triethylammonium) salt 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3, 3,3 ', 3'-tetramethyl-5,5'-disulfoindodicarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetrasodium salt of 5- [4-aza-5-oxo-10- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindodicarbocyanin-1-yl) -dec-1-en-1-yl] -2'-deoxyuridin-5'-triphosphate, tetra potassium salt of 5- [4-aza-5-oxo-10- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindodicarbocyanin-1-yl) -dec-1-en-1-yl] -2'-deoxyuridin-5'-triphosphate, tetralithium salt of 5- [4-aza-5-oxo-10- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindodicarbocyanin-1-yl) -dec-1-en-1-yl] -2'-deoxyuridin-5'-triphosphate, tetraammonium salt of 5- [4-aza-5-oxo-10- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindodicarbocyanin-1-yl) -dec-1-en-1-yl] -2'-deoxyuridin-5'-triphosphate, tetrakis (triethylammonium) salt 5- [4-aza-5-oxo-10- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindodicarbocyanine-1 -yl) -dec-1-en-1-yl] -2'-deoxyuridin-5'-triphosphate, tetrasodium salt of 5- [4,11-diaza-5,12-dioxo-17- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindodicarbocyanine - 1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridin-5'-triphosphate, 5- [4,11-diaza-5,12-dioxo-17- (1 '- (3-trimethylammonio) propyl) -3,3,3' tetra-potassium salt, 3'-tetramethyl-5,5'-disulfoindodicarbocyanine - 1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridin-5'-triphosphate, tetralithium salt of 5- [4,11-diaza-5,12-dioxo-17- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindodicarbocyanine- 1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridin-5'-triphosphate, tetraammonium salt of 5- [4,11-diaza-5,12-dioxo-17- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindodicarbocyanine - 1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridin-5'-triphosphate, tetrakis (triethylammonium) salt 5- [4,11-diaza-5,12-dioxo-17- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl-5,5 ' -disulfoindodicarbocyanin-1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetrasodium salt of 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl-5.5 '-disulfoindodicarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (3-trimethylammonio) propyl) tetra potassium salt -3,3,3', 3'-tetramethyl-5.5 '-disulfoindodicarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetralithium salt of 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl-5.5 '-disulfoindodicarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (3-trimethylammonio) propyl) tetraammonium salt propyl) -3,3,3', 3'-tetramethyl-5.5 '-disulfoindodicarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetrakis (triethylammonium) salt 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl- 5,5'-disulfoindodicarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetrasodium salt of 5- [4-aza-5-oxo-10- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindodicarbocyanin-1-yl) -dec-1-en-1-yl] -2'-deoxyuridin-5'-triphosphate, tetra potassium salt of 5- [4-aza-5-oxo-10- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindodicarbocyanin-1-yl) -dec-1-en-1-yl] -2'-deoxyuridin-5'-triphosphate, tetralithium salt 5- [4-aza-5-oxo-10- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindodicarbocyanin-1-yl) -dec-1-en-1-yl] -2'-deoxyuridin-5'-triphosphate, tetraammonium salt of 5- [4-aza-5-oxo-10- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindodicarbocyanin-1-yl) -dec-1-en-1-yl] -2'-deoxyuridin-5'-triphosphate, tetrakis (triethylammonium) salt 5- [4-aza-5-oxo-10- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindodicarbocyanin-1 -yl) -dec-1-en-1-yl] -2'-deoxyuridin-5'-triphosphate, tetrasodium salt of 5- [4,11-diaza-5,12-dioxo-17- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindodicarbocyanine - 1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridin-5'-triphosphate, 5- [4,11-diaza-5,12-dioxo-17- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindodicarbocyanine tetra potassium salt 1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridin-5'-triphosphate, tetralithium salt of 5- [4,11-diaza-5,12-dioxo-17- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindodicarbocyanine- 1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridin-5'-triphosphate, 5- [4,11-diaza-5,12-dioxo-17- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindodicarbocyanine tetraammonium salt 1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridin-5'-triphosphate, tetrakis (triethylammonium) salt 5- [4,11-diaza-5,12-dioxo-17- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5,5 ' -disulfoindodicarbocyanin-1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetrasodium salt of 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5.5 '-disulfoindodicarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetra potassium salt of 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5.5 '-disulfoindodicarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetralithium salt of 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5.5 '-disulfoindodicarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-tetraammonium salt '-disulfoindodicarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetrakis (triethylammonium) salt 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl- 5,5'-disulfoindodicarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate. 3. The compound of General formula

, where Y represents - (CH ₂ ) ₃ -; - (CH ₂ ) ₅ -; - (CH ₂ ) ₅ CONH (CH ₂ ) ₃ -; X represents —CH ₂ -; -CH ₂ CONH (CH ₂ ) ₅ -; -CH ₂ CONH (CH ₂ ) ₅ CONH (CH ₂ ) ₅ -; M ⁺ represents H ⁺ , Na ⁺ , Li ⁺ , K ⁺ , [CH ₃ (CH ₂ ) _n ] _(4-m) NH _m ⁺ (where n = 0-5, m = 0-4). 4. The compounds of claim 3, which are tetrasodium salt of 5- [4-aza-5-oxo-10- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3', 3'-tetramethyl-5 , 5'-disulfoindotricarbocyanin-1-yl) -dec-1-en-1-yl] -2'-deoxyuridine-5'-triphosphate, 5- [4-aza-5-oxo-10- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3', 3'-tetramethyl-tetra potassium salt , 5'-disulfoindotricarbocyanin-1-yl) -dec-1-en-1-yl] -2'-deoxyuridine-5'-triphosphate, tetralithium salt of 5- [4-aza-5-oxo-10- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3', 3'-tetramethyl-5 , 5'-disulfoindotricarbocyanin-1-yl) -dec-1-en-1-yl] -2'-deoxyuridine-5'-triphosphate, 5- [4-aza-5-oxo-10- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3', 3'-tetramethyl-5 tetraammonium salt , 5'-disulfoindotricarbocyanin-1-yl) -dec-1-en-1-yl] -2'-deoxyuridine-5'-triphosphate, tetrakis (triethylammonium) salt 5- [4-aza-5-oxo-10- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3', 3'- tetramethyl-5,5'-disulfoindotricarbocyanin-1-yl) -dec-1-en-1-yl] -2'-deoxyuridine-5'-triphosphate, tetrasodium salt of 5- [4,11-diaza-5,12-dioxo-17- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3', 3 ' -tetramethyl-5,5'-disulfoindotricarbocyanin-1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, 5- [4,11-diaza-5,12-dioxo-17- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3' tetra potassium salt -tetramethyl-5,5'-disulfoindotricarbocyanin-1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetralithium salt of 5- [4,11-diaza-5,12-dioxo-17- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3', 3 ' -tetramethyl-5,5'-disulfoindotricarbocyanin-1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetraammonium salt of 5- [4,11-diaza-5,12-dioxo-17- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3', 3 ' -tetramethyl-5,5'-disulfoindotricarbocyanin-1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetrakis (triethylammonium) salt 5- [4,11-diaza-5,12-dioxo-17- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3' , 3'-tetramethyl-5,5'-disulfoindotricarbocyanin-1-yl) heptadec-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetrasodium salt of 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3 ', 3'-tetramethyl-5,5'-disulfoindotricarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3 tetra potassium salt ', 3'-tetramethyl-5,5'-disulfoindotricarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetralithium salt of 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3 ', 3'-tetramethyl-5,5'-disulfoindotricarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3,3,3 tetraammonium salt ', 3'-tetramethyl-5,5'-disulfoindotricarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetrakis (triethylammonium) salt 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -3, 3,3 ', 3'-tetramethyl-5,5'-disulfoindotricarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetrasodium salt of 5- [4-aza-5-oxo-10- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindotricarbocyanin-1-yl) -dec-1-en-1-yl] -2'-deoxyuridin-5'-triphosphate, tetra potassium salt of 5- [4-aza-5-oxo-10- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindotricarbocyanin-1-yl) -dec-1-en-1-yl] -2'-deoxyuridin-5'-triphosphate, tetralithium salt of 5- [4-aza-5-oxo-10- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindotricarbocyanin-1-yl) -dec-1-en-1-yl] -2'-deoxyuridin-5'-triphosphate, 5- [4-aza-5-oxo-10- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindotricarbocyanin-1-yl tetraammonium salt) -dec-1-en-1-yl] -2'-deoxyuridin-5'-triphosphate, tetrakis (triethylammonium) salt 5- [4-aza-5-oxo-10- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindotricarbocyanine-1 -yl) -dec-1-en-1-yl] -2'-deoxyuridin-5'-triphosphate, tetrasodium salt of 5- [4,11-diaza-5,12-dioxo-17- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindotricarbocyanine - 1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridin-5'-triphosphate, 5- [4,11-diaza-5,12-dioxo-17- (1 '- (3-trimethylammonio) propyl) -3,3,3' tetra potassium salt, 3'-tetramethyl-5,5'-disulfoindotricarbocyanine- 1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridin-5'-triphosphate, tetralithium salt of 5- [4,11-diaza-5,12-dioxo-17- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindotricarbocyanine- 1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridin-5'-triphosphate, tetraammonium salt of 5- [4,11-diaza-5,12-dioxo-17- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindotricarbocyanine- 1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridin-5'-triphosphate, tetrakis (triethylammonium) salt 5- [4,11-diaza-5,12-dioxo-17- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl-5,5 ' -disulfoindotricarbocyanin-1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetrasodium salt of 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl-5.5 '-disulfoindotricarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (3-trimethylammonio) propyl) tetra potassium salt -3,3,3', 3'-tetramethyl-5.5 '-disulfoindotricarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetralithium salt of 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl-5.5 '-disulfoindotricarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (3-trimethylammonio) propyl) tetraammonium salt propyl) -3,3,3', 3'-tetramethyl-5.5 '-disulfoindotricarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetrakis (triethylammonium) salt 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (3-trimethylammonio) propyl) -3,3,3', 3'-tetramethyl- 5,5'-disulfoindotricarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetrasodium salt of 5- [4-aza-5-oxo-10- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindotricarbocyanin-1-yl) -dec-1-en-1-yl] -2'-deoxyuridin-5'-triphosphate, tetra potassium salt of 5- [4-aza-5-oxo-10- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindotricarbocyanin-1-yl) -dec-1-en-1-yl] -2'-deoxyuridin-5'-triphosphate, tetralithium salt of 5- [4-aza-5-oxo-10- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindotricarbocyanin-1-yl) -dec-1-en-1-yl] -2'-deoxyuridin-5'-triphosphate, tetraammonium salt 5- [4-aza-5-oxo-10- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindotricarbocyanin-1-yl) -dec-1-en-1-yl] -2'-deoxyuridin-5'-triphosphate, tetrakis (triethylammonium) salt 5- [4-aza-5-oxo-10- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindotricarbocyanine-1 -yl) -dec-1-en-1-yl] -2'-deoxyuridin-5'-triphosphate, tetrasodium salt of 5- [4,11-diaza-5,12-dioxo-17- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindotricarbocyanine - 1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridin-5'-triphosphate, 5- [4,11-diaza-5,12-dioxo-17- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindotricarbocyanine tetra potassium salt 1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridin-5'-triphosphate, tetralithium salt of 5- [4,11-diaza-5,12-dioxo-17- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindotricarbocyanine - 1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridin-5'-triphosphate, 5- [4,11-diaz-5,12-dioxo-17- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindotricarbocyanine tetraammonium salt 1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridin-5'-triphosphate, tetrakis (triethylammonium) salt 5- [4,11-diaza-5,12-dioxo-17- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5,5 ' -disulfoindotricarbocyanin-1-yl) -heptadec-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetrasodium salt of 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5.5 '-disulfoindotricarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetra potassium salt of 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5.5 '-disulfoindotricarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetralithium salt of 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-5.5 '-disulfoindotricarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl-tetraammonium salt '-disulfoindotricarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate, tetrakis (triethylammonium) salt 5- [4,11,18-triase-5,12,19-trioxo-24- (1 '- (5-trimethylammonio) pentyl) -3,3,3', 3'-tetramethyl- 5,5'-disulfoindotricarbocyanin-1-yl) -tetracos-1-en-1-yl)] - 2'-deoxyuridine-5'-triphosphate. 5. The compound of General formula intended for the preparation of compounds according to claims. 1 and 2,

, where R is —OH; -NH (CH ₂ ) ₅ COOH; -NH (CH ₂ ) ₅ CONH (CH ₂ ) ₅ COOH. 6. The compounds of claim 5, which are 1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -1- (5-carboxypentyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindodicarbocyanine dye, N- (7-aza-12-carboxy-6-oxododec-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (7-aza-10-trimethylammonio-6-oxodec-1- sludge) -5,5'-disulfoindodicarbocyanine dye, N- (7,14-diaza-19-carboxy-6,13-dioxononadec-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (7-aza-10-trimethylammonio-6- oxodec-1-yl) -5,5'-disulfoindodicarbocyanine dye. 7. The compound of General formula, intended for the preparation of compounds according to claims. 1 and 2,

, where R is —NH (CH ₂ ) ₅ COOH; -NH (CH ₂ ) ₅ CONH (CH ₂ ) ₅ COOH (m = 3); -HE; -NH (CH ₂ ) ₅ COOH; -NH (CH ₂ ) ₅ CONH (CH ₂ ) ₅ COOH (m = 5). 8. The compounds of claim 7, which are N- (7-aza-12-carboxy-6-oxododec-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (3-trimethylammoniopropyl) -5,5'-disulfoindodicarbocyanine dye, N- (7,14-diaza-19-carboxy-6,13-dioxononadec-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (3-trimethylammoniopropyl) -5,5'- disulfoindodicarbocyanine dye, N- (5-carboxypentyl) -3,3,3 ', 3'-tetramethyl-N' - (5-trimethylammoniopentyl) -5,5'-disulfoindodicarbocyanine dye, N- (7-aza-12-carboxy-6-oxododec-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (3-trimethylammoniopentyl) -5,5'-disulfoindodicarbocyanine dye, N- (7,14-diaza-19-carboxy-6,13-dioxononadec-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (3-trimethylammoniopentyl) -5,5'- disulfoindodicarbocyanine dye. 9. The compound of General formula, intended for the preparation of compounds according to claims. 3 and 4,

, where R is —OH; -NH (CH ₂ ) ₅ COOH; -NH (CH ₂ ) ₅ CONH (CH ₂ ) ₅ COOH. 10. The compounds of claim 9, which are 1 '- (7-aza-10-trimethylammonio-6-oxodec-1-yl) -1- (5-carboxypentyl) -3,3,3', 3'-tetramethyl-5,5'-disulfoindotricarbocyanine dye, N- (7-aza-12-carboxy-6-oxododec-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (7-aza-10-trimethylammonio-6-oxodec-1- sludge) -5,5'-disulfoindotricarbocyanine dye, N- (7,14-diaza-19-carboxy-6,13-dioxononadec-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (7-aza-10-trimethylammonio-6- oxodec-1-yl) -5,5'-disulfoindotricarbocyanine dye. 11. The compound of General formula, intended for the preparation of compounds according to claims. 3 and 4,

, where R is —OH; -NH (CH ₂ ) ₅ COOH; -NH (CH ₂ ) ₅ CONH (CH ₂ ) ₅ COOH; m = 3-5. 12. The compounds of claim 11, which are N- (5-carboxypentyl) -3,3,3 ', 3'-tetramethyl-N' - (3-trimethylammoniopropyl) -5,5'-disulfoindotricarbocyanine dye, N- (7-aza-12-carboxy-6-oxododec-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (3-trimethylammoniopropyl) -5,5'-disulfoindotricarbocyanine dye, N- (7,14-diaza-19-carboxy-6,13-dioxononadec-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (3-trimethylammoniopropyl) -5,5'- disulfoindotricarbocyanine dye, N- (5-carboxypentyl) -3,3,3 ', 3'-tetramethyl-N' - (5-trimethylammoniopentyl) -5,5'-disulfoindotricarbocyanine dye, N- (7-aza-12-carboxy-6-oxododec-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (3-trimethylammoniopentyl) -5,5'-disulfoindotricarbocyanine dye, N- (7,14-diaza-19-carboxy-6,13-dioxononadec-1-yl) -3,3,3 ', 3'-tetramethyl-N' - (3-trimethylammoniopentyl) -5,5'- disulfoindotricarbocyanine dye. 13. The use of compounds according to claims. 1-4 as fluorescent labels for enzymatic DNA labeling during PCR.

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