RU2637310C1 - Fluorescently-labeled deoxyuridine triphosphates - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: invention relates to fluorescently-labeled deoxyuridine triphosphates, which can be used as labels for DNA labeling during PCR. The compounds of the invention include 5-[4-aza-5-oxo-9-(3,3',3'-trimethyl-1-(4-sulfonatobutyl)-1'-ethylindodicarbocyanin-3-yl)-non-1-en-1-yl)]-2'-deoxyuridine-5'-triphosphate, 5-[4-aza-5-oxo-9-(3,3',3'-trimethyl-1,1'-di-(4-sulfonatobutyl)indodicarbocyanin-3-yl)-non-1-en-1-yl)]-2'-deoxyuridine-5'-triphosphate, 5-[4-aza-5-oxo-9-(3,3',3'-trimethyl-1'-(4-sulfonato-butyl)-1-ethylindodicarbocyanin-3-yl)-non-1-en-1-yl)]-2'-deoxyuridine-5'-triphosphate, 5-[4-aza-5-oxo-9-(3,3',3'-trimethyl-5'-sulfo-1,1'-diethylindodicarbocyanin-3-yl)-non-1-en-1-yl)]-2'-deoxyuridine-5'-triphosphate, 5-[4-aza-5-oxo-9-(3,3',3'-trimethyl-1,1'-diethyl-5-sulfo-indodicarbocyanin-3-yl)-non-1-en-1-yl)]-2'-deoxyuridine-5'-triphosphate, 5-[4-aza-5-oxo-7-(1-(4 3-tetramethyl-1'-ethylindodicarbocyanin-5-yl)-hept-1-en-1-yl)]-2'-deoxyuridine-5'-triphosphate, 5-[4-aza-5-oxo-7-(1'-(4-sulfonato-butyl)-3,3,3',3'-tetramethyl-1-ethylindodicarbocyanin-5-yl)-hept-1-en-1-yl)]-2'-deoxyuridine-5'-triphosphate, 5-[4-aza-5-oxo-7-(3,3,3',3'-tetramethyl-5'-sulfo-1,1'-diethylindodicarbocyanin-5-yl)-hept-1-en-1-yl]-2'-deoxyuridine-5'-triphosphate or their salts. New fluorescently-labeled nucleotides are proposed that are effective as fluorescent labels for DNA labeling during PCR, including application of the known test systems to identify mutations associated with drug resistance to M. tuberculosis and with a genetic predisposition to thrombosis.

EFFECT: increased efficiency and sustainability.

3 cl, 13 dwg, 4 tbl, 45 ex

Description

FIELD OF THE INVENTION

The invention relates to the field of nucleic acid chemistry, and in particular to new modified nucleotides - fluorescently-labeled deoxyuridine triphosphates. The invention includes the synthesis of nucleotides modified at the C5 position of the pyrimidine ring, as well as their use for the analysis of nucleic acids (DNA).

State of the art

Nucleic acid analysis is used both for conducting a wide range of studies in various fields of science, as well as for clinical diagnostics, pharmacology, medicine, biotechnology, etc. The introduction of markers visualizing synthesized DNA allows us to study the interaction of the studied DNA with various targets: DNA, RNA, proteins, low molecular weight compounds. Fluorescence labeling of nucleic acids is one of the ways to identify the results of hybridization analysis, in particular, in the technology of biological microarrays, in microscopic cytometry, in flow cytometric sorting, etc. In all of these methods, the analyzed DNA is initially detected using a complementary nucleic acid. Then, the amount of duplexes formed is measured by the fluorescence intensity of the label associated with one of the duplex components.

The introduction of fluorescently labeled nucleotides using DNA polymerases has been widely used for labeling genes and DNA. Samples with a high density of tags can increase the sensitivity of the analysis. Polymerase chain reaction is a convenient method for labeling and amplification of DNA with a high density of fluorophores with the formation of fluorescently labeled nucleotides, which are substrates for DNA polymerases during PCR. However, the use of fluorescently labeled nucleotides has some limitations, since most enzymes do not recognize modified nucleotides poorly. Labeling efficiency is also highly dependent on the type of dye in the modified nucleotides.

It is known that the C5 position of the pyrimidine cycle is best suited for nucleotide modification, since it is oriented into a wide groove of double-stranded DNA and does not inhibit the interaction of AT bases. Functional markers such as fluorophores, enzymes, ligands, for example biotin, are attached to the C5 position of the pyrimidine base via an amino linker (Tester J.K. et al., Synthesis and characterization of DNA oligomers and duplexes containing covalently attached molecular labels: comparison of biotin, fluorescein, and pyrene labels by thermodynamic and optical spectroscopic measurements, J. Am. Chem. Soc., 1989, vol. 111, pp. 6966-6976). Often, modified DNAs are prepared using DNA synthesizers (Ketomaki K. et al., Hybridization properties of support-bound oligonucleotides: the effect of the site of immobilization on the stability and selectivity of duplex formation, Bioconjugate Chem., 2003, vol. 14 , pp. 811-816).

However, while DNA polymerases can accept modified nucleotides as substrates, modified DNAs are produced enzymatically. The enzymatic production of modified DNA, namely labeled with fluorophores, is of particular interest. The combination of polymerase chain reaction (PCR), which allows you to get many copies of the studied DNA, with simultaneous fluorescence labeling is extremely important.

For PCR, a mixture of deoxynucleoside triphosphates is used, which contains dATP, dGTP, dCTP and dTTP, and dUTP or a mixture of dTTP / dUTP in any molar proportion can be used instead of dTTP. Any of these deoxynucleoside triphosphates may carry a fluorescent label (US Pat. No. 6,589,737, Compositions and methods for labeling of nucleic acid molecules, 07/08/2003). Most preferably, the use of fluorescently-labeled deoxyuridine triphosphate, which, on the one hand, determines the effective incorporation of this substrate into the newly synthesized DNA chain during PCR, on the other hand, makes it possible to prevent false results due to cross-contamination with amplicons by using enzyme in the first stage uracil DNA glycosylase.

The efficiency of incorporation into DNA of derivatives bearing substituents at the C5 position of the pyrimidine cycle depends on the nucleotide, the nature of the substituent, and the type of DNA polymerase (Kuwahara M. et al., Systematic characterization of 2'-deoxynucleoside-5'-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on enzymatic production of modified DNA, Nucleic Acids Res., 2006, vol. 34, No. 19, pp. 5383-5394).

Derivatives of 2'-deoxyuridine with a propynyl group in the C5 position are good substrates for Vent and Taq DNA polymerases (Giller G. et al., Incorporation of reporter molecule-labeled nucleotides by DNA polymerases. I. Chemical synthesis of various reporter group- labeled 2'-deoxyribonucleoside-5'-triphosphates, Nucleic Acids Res. 2003, vol. 31, No. 10, pp. 2630-2635). Derivatives with a propenyl group are good substrates for Taq-DNA polymerase, but poor for Vent- and Pfu-DNA polymerases, and with a propyl group very poor substrates for Taq-DNA polymerase (Godovikova TS et al., 5- [3- (E) - (4-azido-2,3,5,6-tetrafluorobenzamido) propenyl-1] -2'-deoxyuridine-5'-triphosphate substitutes for thymidine-5'-triphosphate in the polymerase chain reaction, Bioconjugate Chem., 1999, vol. 10, pp. 529-537; Gourlain T. et al, Enhancing the catalytic repertoire of nucleic acids. II. Simultaneous incorporation of amino and imidazolyl functionalities by two modified triphosphates during PCR, Nucleic Acids Res., 2001, vol. 29 No. 9, pp. 1898-1905). In other words, for 2'-deoxyuridine, good substrates are derivatives with hard linkers, linkers based on ethine (acetylene) and E-ethene (ethylene, transisomers), and bad ones with flexible linkers, linkers based on ethane and Z-ethene (cis- isomers). For 2'-deoxycytidine, propynyl C5 derivatives are good substrates for Vent and Taq DNA polymerases.

Even a slight chemical modification of the amino group of 5-aminoallyl-2'-deoxyuridine-5'-triphosphate leads to a significant difference in the incorporation of DNA polymerases into the PCR product. However, the effectiveness of enzymatic inclusion depends not only on the substituent located in the immediate vicinity of the pyrimidine ring position, but also on the more distant environment. In the publication (Giller G. et al., Incorporation of reporter molecule-labeled nucleotides by DNA polymerases. I. Chemical synthesis of various reporter group-labeled 2'-deoxyribonucleoside-5'-triphosphates, Nucleic Acids Res. 2003, vol. 31 , No. 10, pp. 2630-2635) showed that the incorporation efficiency of fluorescently-labeled derivatives of 5-aminoallyl-2'-deoxyuridine-5'-triphosphate Vent (exo) -HHK polymerase significantly depends on the nature of the fluorophore attached to the amino group of the linker.

As the fluorescent label, any fluorescent dye can be used that can be chemically introduced into the deoxynucleoside triphosphate molecule in such a way as to not substantially interfere with the passage of the polymerase chain reaction. In the case of fluorescently-labeled deoxyuridine triphosphate, for example, a fluorescent dye can be attached to the 5'-end of the aminopropargyl derivative dUTP (Ranasinghe RT et al, Fluorescence based strategies for genetic analysis, Chem. Commun, 2005, pp. 5487-5502). Dyes of fluorescein (TAMRA ^® , ROX ^® , JOE ^® ), rhodamine (Texas Red ®), indocyanine (Cy3 ^® , Cy5 ^® , Cy5.5 ^® , Cy7 ^® ) series are used. The most preferred are dyes, the excitation spectrum of which lies in the long-wave (red) region of the spectrum with lower background fluorescence, the region of the “biological tissue diagnostic window”, which allows increasing the sensitivity of the analysis.

Indocyanine dyes are widely used to obtain fluorescently-labeled DNA. The nature of the substituents and the sites of their binding to the fluorophore core of the dye affect the brightness of fluorescence, solubility, and the formation of molecular aggregates of both the dye itself and the nucleic acids labeled by it. The orientation of the fluorophore dye core relative to the marked nucleotide, the length and chemical nature of the linker that moves the fluorophore from the marked DNA significantly affect the efficiency of the formation of perfect duplexes with complementary DNA, and ultimately affect the sensitivity of bioanalysis in general. Indocyanines containing various substituents — alkyl, sulfo, sulfoalkyl, condensed aryl, have long been known. Each new series of indocyanine dyes serves as the basis for obtaining a new series of fluorescently-labeled biologically active compounds and intermediates for their preparation.

In the publication (European Patent No. 1152008, Fluorescent nucleotides containing a cyanine, merocyanine or styryl dye for the detection of nucleic acid, 11/07/2001), fluorescently-labeled 5-aminoallyl deoxyurid triphosphates containing indocarbo and indodicarbocyanine dyes (various dyes sulfamide, chlorine, hydroxyl).

The publication (Yu N. et al., Cyanine dye dUTP analogs for enzymatic labeling of DNA probes, Nucleic Acids Res., 1994, vol. 22, No. 15, pp. 3226-3232) presents a synthesis of Cy5-dUTP and Cy3-dUTP used to obtain fluorescently labeled nucleic acid fragments using nick translation methods, random prime labeling and PCR.

The publication (Lacenere CJ et al, Effects of a modified dye-labeled nucleotide spacer arm on incorporation by thermophilic DNA polymerases, Nucleosides, Nucleotides and Nucleic Acids, 2006, vol. 25, pp. 9-15) revealed the influence of the length of the linker connecting fluorophore and AAdUTP, on the growth efficiency of the chain of synthetic primers depending on the DNA polymerases used. It was shown that a long linker leads to a more efficient elongation, and products are formed longer than the initial primer by 30-52 base pairs, while a short linker promotes the formation of products longer by 5-10 base pairs.

The publication (European Patent No. 1211294, Improved process and method for the preparation of asymmetric monofunctionalised indocyanine labelling reagents and obtained compounds, 06/05/2002) presents the synthesis of fluorescently-labeled sulfobenzindocyanine dyes of deoxyuridine triphosphates. In the publication (European Patent No. 1801165, Modified carbocyanine dyes and their conjugates, 06/27/2007), Molecular Probes proposed fluorescently labeled 5-aminoallyl and 5-aminopropargyldeoxyuridine triphosphates containing polysulfonated indocyanine dyes with a carboxypentine 3 position substituent . The synthesized compounds are recommended for nick translation and in-vitro transcription.

In the publication (European Patent No. 1491591, New fluorescence cyanine labels containing a sulfamido linker arm, 12/29/2004) a general procedure is proposed for the preparation of fluorescently labeled 5-aminopropargyldeoxyuridine and deoxycytidine triphosphates based on hydroxysuccinimide esters of cyanine dyes with a carboxymethyl sulfonyl group, position 5, with a different number of methylene units from 1 to 6. The authors argue that such dyes have a distinct advantage over their analogues - with a carboxyalkyl group in positions 1 and 5. Priso union of the carboxyl function via a sulfamide bond led to increased stiffness of the molecule, and because of this, and to improve the characteristics of the fluorescent dye.

In a publication (US Patent No. 2004186278, Cyanine dye labelling reagents with meso-substitution, September 23, 2004), a cyanine dye with a nitrile substituent in the meso position of the polymethine chain was used to obtain fluorescently labeled deoxyuridine triphosphate.

The approach to assessing the effectiveness of the enzymatic incorporation of fluorescently labeled nucleotide derivatives using oligonucleotide biochips has not been described in the literature and seems very effective. The ability to simultaneously observe several tens of signals under conditions of kinetic and thermodynamic control allows you to simultaneously receive a large amount of quantitative information, with greater reliability. The presence of a large number of examples of fluorescently labeled derivatives of 2'-deoxyuridine-5'-triphosphate and a quantitative assessment on the developed models of biochips will clearly determine the effect of various structural factors on the efficiency of incorporation into the PCR product.

Disclosure of invention

The aim of the invention was the synthesis of nucleotide derivatives containing a cyanine dye in position C5, as well as their use for DNA labeling during PCR. The resulting modified DNA acquires new properties due to the introduced modifications, but must specifically interact with DNA and proteins. Our approach is based on the synthesis of indocyanine dyes containing two heterocyclic ring systems linked by a polymethine linker linker and containing a carboxyl group at position 3 or 5 of the indolenine heterocycle attached via a polymethylene chain. Such compounds proposed in this invention are compounds of General formula (I).

where R ¹ represents-CH ₃ ; - (CH ₂ ) ₄ COOH;

R ² represents - (CH ₂ ) ₂ COOH; -H; -SO ₃ ^- ;

R ³ represents —C ₂ H ₅ ; - (CH ₂ ) ₄ SO ₃ ^- ;

R ⁴ represents —H; -SO ₃ ^- ;

R ⁵ represents —C ₂ H ₅ ; - (CH ₂ ) ₄ SO ₃ ^- .

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

where R ¹ represents -H; -SO ₃ ^- ;

R ² represents —C ₂ H ₅ ;-( CH ₂ ) ₄ SO ₃ ^- ;

R ³ represents —H; -SO ₃ ^- ;

R ⁴ represents —C ₂ H ₅ ; - (CH ₂ ) ₄ SO ₃ ^- .

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

where R ¹ represents-C ₂ H ₅ ; - (CH ₂ ) ₄ SO ₃ ^- ;

R ² represents —H; -SO ₃ ^- ;

R ³ represents —C ₂ H ₅ ; - (CH ₂ ) ₄ SO ₃ ^- .

The proposed fluorescently-labeled triphosphates of the general formula (TA) are characterized in that the reactive carboxyl group of the indocyanine dyes of the general formula (A) included in their composition is attached at the 3 position of the indolenin fragment through a tetramethylene chain of atoms (where R ¹ , R ² , R ³ , R ⁴ are alkyl, sulfo and sulfoalkyl substituents).

The proposed fluorescently-labeled triphosphates of the general formula (TB) are characterized by the fact that the reactive carboxyl group of the indocyanine dyes of the general formula (B) included in their composition is attached at the 5 position of the indolenin fragment through a dimethylene chain of atoms (where R ¹ , R ² , R ³ - alkyl, sulfo and sulfoalkyl substituents).

The indicated structural differences of the indocyanine dyes of the general formulas (A) and (B), which are part of the nucleoside triphosphates (TA) and (TB), lead to a number of distinctive properties, namely, increased light resistance, increased chemical stability, and heat resistance when used as fluorescent labels .

It was shown that during continuous irradiation with a fluorescence microscope at a wavelength of 655 nm, the half-life of dyes of formulas (A) and (B) differs: for dye (A2), the half-life is 7 hours, for dyes (Al), (B1) - 5, 3 hours, the dye (A3), (B2) - 6 hours for the dye (A4) - 6.9 hours, while the dye constituting the triphosphate of the general formula (I) (R ¹ = -CH ₃ ; R ² = R ⁴ = -H; R ³ = - (CH ₂ ) ₅ COOH, R ⁵ = -C ₂ H ₅ ) is 5.5 hours (Fig. 1 line No. 4), for the dye of formula (I) (R ¹ = —CH ₃ ; R ² = R ⁵ = —H, R ₃ = - (CH ₂ ) ₅ COOH, R ⁴ = - (CH ₂ ) ₄ SO ₃ ^- ) is 1.5 hours (Fig. 1 line No. 6), for the dye of formula (I) (R ¹ = -CH ₃ ; R ² = -SO ₃ ^- , R ³ = - (CH ₂ ) ₅ COOH, R ⁴ = - (CH ₂ ) ₄ SO ₃ ^- ) is 2.5 hours (Fig. 1 line No. 7 )

The values of the molar extinction coefficient of dyes (A1) and (A2) exceed 240,000, (B1) - about 200,000 (table 1, Fig. 2). The quantum yield of fluorescence of dyes (A1) - (A4) in methanol exceeds 25% (table 1, Fig. 3).

The relative fluorescence efficiency of the dyes of the general formulas (A) and (B) was determined, while the highest efficiency value was obtained for the dye (A2) upon excitation and registration at the absorption and fluorescence maxima and was taken as unity (table 1, Fig. 4). The following data were obtained:

- upon excitation and recording at the maxima of absorption and fluorescence, the relative fluorescence efficiency exceeds 0.6 for dyes (A1) - (A4) in methanol and for dye (A2) in PBS;

- when excited at a wavelength of 655 nm and recorded at 690 nm, the relative fluorescence efficiency exceeds 0.2 for the dye (A2) in methanol and more than 0.1 in PBS.

The proposed dyes of structures (A) and (B), depending on the substituents in the heterocyclic fragment, differ in the spectral ranges of excitation and fluorescence, the total electric charge and solubility in water (table 1). Due to the spectral characteristics in the near infrared range, indodicarbocyanine dyes of formulas (A) and (B) are optimal for labeling biomolecules, since the biomaterial in the wavelength range between 600-800 nm has low intrinsic fluorescence. The introduction of labels into DNA in the form of fluorescently labeled nucleoside triphosphates of the general formula I at the stage of cDNA production by PCR allows for higher sensitivity, since the marker is introduced not in the singular, but in greater quantity together with nucleotides (Yu H. et al., Cyanine dye dUTP analogs for enzymatic labeling of DNA probes, Nucleic Acids Res., 1994, vol. 22, No. 15, pp. 3226-3232).

The following are the results of the use of dyes to obtain fluorescently labeled nucleoside triphosphates, followed by their use for DNA labeling during PCR. All dyes of the general formulas (A1) - (A5) and (B1) - (B3) as their N-hydroxysuccinimide esters (for (A) R ₁ = - (CH ₂ ) ₄ COOSu; for (B) R ₂ = - (CH _{2) 2} COOSu) and p-nitrophenyl esters (for (a) R ₁ = - (CH _{2) 4} COOpNP; for (B) R ₂ = - (CH _{2) 2} COOpNP) in good yield labeled nucleotides, comprising in the C5 position, an aminoallyl substituent.

The spectral properties of fluorescently labeled nucleotides of the general formula I are presented in Table 2. Modified nucleotides are characterized by intense absorption and fluorescence bands (Fig. 5) and fluorescence quantum yield values of 15-20% in phosphate-buffered saline (Fig. 6). Compared with dyes (A) and (B), a bathochromic shift of the absorption and fluorescence maxima by several nm is observed, as well as an increase in the quantum by 1-7%, depending on the structure of the compound. The absorption and fluorescence maxima of the modified nucleotides I are located at 645 nm and 663 nm, respectively. We observed a bathochromic shift in the maximum absorption of uridine-5'-triphosphate from 262 nm to ~ 300 nm when it was modified by the addition of an aminoalkyl substituent bearing a fluorescent label.

It was found that the results of the polymerase chain reaction using fluorescently labeled nucleoside triphosphates of the general formula I (TA1) - (TA5) and (TB1) - (TB3) depend on the structure of the modified nucleotides used as substrates in PCR. For a comparative assessment of the degree of incorporation of modified nucleotides, the average fluorescence signal of perfect duplexes in the biochip cells is calculated and then normalized to the value obtained for a commercially available product manufactured by Amersham (CY5-DUTP, Amersham PA55022). It was found that the formation of a full-sized PCR product depends not only on the chemical structure of the substituents, but also on the location of the substituent in the fluorophore molecule. The fluorescent signal is higher for nucleotides (TA1), (TA3) - (TA5) and (TB1) - (TB3) containing a single sulfo group cyanine dye (Fig. 7, Fig. 11). The fluorescent signals obtained for nucleotide (TA2) and commercially available Cy5-dUTP, were lower and approximately equal. It was found that the location of the sulfo group in the dye molecule affects the efficiency of incorporation of modified nucleotides into the PCR product.

It was found that all dyes of the formulas (A1) - (A5) and (B1) - (B3) in the conjugates with nucleotides do not inhibit the polymerase chain reaction (Fig. 9, Fig. 10, Fig. 12). Modified nucleotides of the general formula (I) do not affect the stability of all other components of the analysis. It was found that all compounds of formula (I) (TA1) - (TA5) and (TB1) - (TB3) are stable enough to conduct a complete analysis cycle without noticeable dye decomposition. By the combination of all the properties of the compounds of formula (I) (TA1) - (TA5) and (TB1) - (TB3) they can be used as fluorescent labels in the development of diagnostic test systems based on PCR.

Brief Description of the Drawings

FIG. 1. The dependence of the fluorescence intensity on time when the dyes on the biochip are irradiated with a semiconductor laser light source (655 nm / 690 nm, 40 mW): 1 - dye (A2); 2 - dye (A4); 3 - dyes (A3, B2); 4 - a colorant of formula (I) (R ¹ = —CH ₃ ; R ² = R ⁴ = —H; R ³ = - (CH ₂ ) ₅ COOH, R ⁵ = —C ₂ H ₅ ); 5 - dyes (A1, B1); 6 - dye of the formula (I) (R ¹ = —CH ₃ ; R ² = R ⁵ = —H, R ₃ = - (CH ₂ ) ₅ COOH, R ⁴ = - (CH ₂ ) ₄ SO ₃ ^- ); 7 is a dye of formula (I) (R ¹ = —CH ₃ ; R ² = —SO ₃ ^- , R ³ = - (CH ₂ ) ₅ COOH, R ⁴ = - (CH ₂ ) ₄ SO ₃ ^- ).

FIG. 2. Coefficients of molar extinction of dyes in methanol and PBS.

FIG. 3. Quantum yield of fluorescence of dyes in methanol and PBS.

FIG. 4. Relative fluorescence efficiency of dyes: excitation - at the absorption maximum and at a wavelength of 655 nm, registration - at the fluorescence maximum and at a wavelength of 690 nm, respectively.

FIG. 5. Normalized absorption and fluorescence spectra of fluorescently-labeled deoxyuridine triphosphate.

FIG. 6. Quantum yield of fluorescence of modified deoxyuridine triphosphates in PBS.

FIG. 7. DNA hybridization of a wild-type sample on a TB biochip.

FIG. 8. Electrophoretic analysis in a 2% agarose gel of the results of multiplex amplification of the rpoB, katG, inhA, ahpC genes and the insertion element IS6110 with various fluorescently-labeled deoxyuridine triphosphates in the reaction mixture at a concentration of 8 μM: (a) manifestation of ethidium bromide, (b) manifestation light fluorescence of cyanine dye. Lane 1 - molecular weight marker pUC19 / MspI (67-501 bp); lane 2 - positive control, does not contain labeled nucleoside triphosphate; lane 3 - nucleotide TA2; lane 4 - nucleotide TA1; lane 5 - nucleotide TA3; lane 6 - nucleotide TA4; lane 7 - nucleotide TA5; lane 8 - nucleotide TBI; lane 9 - nucleotide TB2; lane 10 - nucleotide TB3; lane 11 - negative amplification control.

FIG. 9. The dependence of the normalized fluorescence signal on the concentration of fluorescently-labeled nucleoside triphosphate TA2 and TA4.

FIG. 10. Normalized fluorescence signal for various modified nucleotides when using the TB-biochip diagnostic test system.

FIG. 11. Hybridization, normalized signal, and genotype of labeled DNA fragments of samples on a thrombo-biochip using the example of fluorescently labeled nucleotide TA1 (wt - wild type, mut - mutation).

FIG. 12. Electrophoretic analysis in a 2% agarose gel of the results of multiplex amplification of genes of the fibrinolysis system: (a) the manifestation of ethidium bromide, (b) the manifestation in light of the fluorescence of the cyanine dye. A - FGB gene, B - F7 gene, C-MTHFR gene, D - PAI-1 gene, E - GPIIIa gene. Lane 1 - nucleotide TA2; lane 2 - nucleotide TA1; lane 3 — TA3 nucleotide; lane 4 — TA4 nucleotide; lane 5 - nucleotide TA5; lane 6 - nucleotide TBI; lane 7 - nucleotide TB2; lane 8 — nucleotide TB3; lane 9 - positive control, does not contain labeled nucleoside triphosphate; lane 10 - negative amplification control; lane 11 — molecular weight marker pUC19 / MspI (67-501 bp).

FIG. 13. The normalized fluorescence signal for various modified nucleotides using the diagnostic test system "PF-biochip Trombo."

The implementation of the invention

The compounds of the invention of the general formulas TA and TB were synthesized according to Scheme 1.

Scheme 1

The synthesis of asymmetric indodicarbocyanine dyes of formulas (A, B) that are part of TA and TB nucleoside triphosphates is based on the condensation reaction of indoleninium salts containing an active methylene group and malany dialdehyde dianyl hydrochloride (Scheme 2, Table 3, 4) (B.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, p. 2355- 2359; VE Kuznetsova et al., Mend. Comm, 2008, No. 3, pp. 138-140). The synthesis of intermediate compounds necessary for the preparation of dyes of the formulas (A, B) is presented in schemes 3–9 (V.E. Kuznetsova et al. Izv. AN, Ser. Chem. - 2008. - No. 3. - P. 595- 598; V.E. Kuznetsova et al. Izv. AN, Ser.chem., 2007, No. 11, pp. 2186-2190).

Scheme 2

Scheme 3

Scheme 4

Scheme 5

Scheme 6

Scheme 7

Scheme 8

Scheme 9

The starting compound for the synthesis of indolenins (7) (Scheme 4) and (9) (Scheme 5) containing a functional group at position 3 is 6-methyl-7-oxo-octanoic acid (6), the synthesis of which is shown in Scheme 3. Acetylation of cyclohexanone carried out in two ways: a complex of BF ₃ and ⋅2AcOH action of acetyl chloride in chloroform at morpholino cyclohexene derivative (2) in the presence of Et ₃ N. 2-Acetyl-2-methylcyclohexanone (4) was obtained in the action of methyl iodide in 2-atsetiltsiklogeksanona solution (3) methyl alcohol. Then, keto acid methyl ester was synthesized using lithium methylate (5). The hydrolysis of which was carried out with dilute hydrochloric acid, while obtaining keto acid (6). The condensation of phenylhydrazines and 6-methyl-7-oxo-octanoic acid (6) (Scheme 3) gave the corresponding indolenine bases (7, 9) (Scheme 4, 5).

The starting compound for the synthesis of indolenin (17) containing a functional group at position 5 is 4- (2-carboxyethyl) phenylhydrazine hydrochloride (16) (Scheme 6). Nitrile (12) was obtained from 4-nitroaniline by the action of a solution of acrylonitrile in acetone on the diazonium salt formed during the reaction. 3- (4-Nitrophenyl) acrylonitrile (13) was obtained by boiling a water-ethanol solution of 2-chloro-3- (4-nitrophenyl) propionitrile (12) in the presence of sodium acetate. 3- (4-Nitrophenyl) -2-propenoic acid (14) was obtained by acid hydrolysis of nitrile (13). Further reduction of the acid (14) led to the production of 3- (4-aminophenyl) propionic acid (15). 4- (2-Carboxyethyl) phenylhydrazine hydrochloride (16) was obtained by reduction of the diazonium salt in the acidic medium, which was formed during the reaction. The carboxyethyl derivative of indolenin (17) was synthesized from hydrazine (16) and methyl isopropyl ketone in glacial acetic acid in the presence of potassium acetate without isolating the hydrazone formed during the reaction.

The condensation of phenylhydrazine (1) with methylisopropyl ketone gave the corresponding indolenine (20) (Scheme 7).

N-sulfoalkylation of all indolenin bases was carried out in 1,2-dichlorobenzene or butyronitiril under the action of 1,4-butanesultone (compound (11) scheme 5, compound (19) scheme 6, compound (22) scheme 8). N-Alkyl derivatives were obtained by the action of n-toluenesulfonic acid ethyl iodide or ethyl ester on a solution of indolenine in acetoniryl or butyronitrile (compound (8) scheme 4, compound (10) scheme 5, compound (18) scheme 6, compound (21) scheme 7, compound (23) Scheme 8).

Polymethine bridge was prepared as follows (Scheme 9). The reaction of ethyl vinyl ether and triethylorthoformate in the presence of boron trifluoride etherate gave malonic aldehyde tetraethyl acetal (24). Further, under the action of aniline hydrochloride, malonic dialdehyde dianyl hydrochloride was obtained (25).

Asymmetric indodicarbocyanine dyes of formulas (A, B) are synthesized by condensation of indoleninium salts and malany dialdehyde dianyl hydrochloride (Scheme 2, tables 3, 4). In the course of the reaction, in addition to the target compound, two more symmetrical by-products are formed. In the first stage, indolenine alone was introduced into the condensation reaction. The progress of the reaction was monitored spectrophotometrically, with the disappearance of the λ _max 237 and 280 nm peaks characteristic of indolenins, and the appearance of a new λ _max 440 nm signal characteristic of the intermediate. For the complete reaction to take place, heating at 118 ° C for 1-3 hours is sufficient. The second stage of obtaining the indodicarbocyanine dye is carried out using diisopropylethylamine or anhydrous potassium acetate as condensing agents. All synthesized indodicarbocyanine dyes were isolated by reverse phase chromatography in acetonitrile-0.01M triethylammonium acetate buffer solution by gradient elution from 0 to 50% acetonitrile. Next, their sodium salts were obtained.

dye p-nitrophenyl esters were obtained by the action of bis p-nitrophenyl carbonate in DMF. Active esters were isolated by reverse phase chromatography. The reaction of the AAdUTP amino group with 1.25 equivalent of p-nitrophenyl ether dye of the general formula (A) or (B) in 0.1 M NaHCO ₃ / Na ₂ CO ₃ (pH 8.5) in DMF yields fluorescently-labeled nucleoside triphosphates of the general formula (I) with a yield of approximately 40% depending on the structure of the dye used (Scheme 1). Fluorescence-labeled nucleotides were purified in 2 stages — anionic ion exchange chromatography on a DEAE-cellulose sorbent and reverse phase (C18-RP) chromatography.

Another object of the invention is the use of the compounds of the invention of general formula (I) for DNA labeling. Enzymatic administration of compounds of formulas (TA) and (TB) is presented using the TB-biochip diagnostic test system as an example to determine mutations associated with drug resistance of M. Tuberculosis.

The procedure for molecular identification of mutations consists of three successive stages: (1) multiplex PCR with amplification of 5 DNA fragments; (2) asymmetric multiplex PCR with the formation of 5 fluorescently-labeled single-stranded DNA fragments; (3) hybridization of labeled fragments with an oligonucleotide microchip (biochip). Fluorescently-labeled nucleoside triphosphates of the general formula (I) were added to a mixture of natural dNTPs used in PCR. During PCR, an amplicon was obtained that contained a fluorescent label. The fluorescently-labeled amplicon was hybridized on a biochip.

The microchip (Fig. 7) 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 structure of the probe does not correspond to the structure of the amplicon, 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.

The formation of full-sized PCR products was confirmed by horizontal agarose gel electrophoresis in the presence of ethidium bromide and using a fluorescence analyzer (Fig. 8). The electrophoretic bands of PCR products (3-10) obtained by both methods are identical. All modified nucleotides of the general formula (I) do not inhibit PCR for the Mycobacterium tuberculosis rpoB gene region and integrate into amplified products.

Hybridization of wild type M. tuberculosis with a biochip is shown in FIG. 7. The intensities of hybridization signals on a biological microchip for PCR products obtained with different fluorescently-labeled deoxyuridine triphosphates are different. Various factors influence the magnitude of the signals: the efficiency of incorporation of modified nucleotides by DNA polymerase into the amplified product, specific binding with the formation of perfect duplexes with oligonucleotide probes located in the biochip cells, non-specific binding to the components of the biochip cell, and the brightness of the fluorescence of the fluorescence tag associated with the amplification.

The optimal concentration of fluorescently-labeled deoxyuridine triphosphates was determined for PCR using Taq DNA polymerase (Fig. 9), which corresponds to 8 μM. In FIG. 10 shows the levels of fluorescence signals on a chip with various fluorescently-labeled deoxyuridine triphosphates at a concentration of 8 μM. The average fluorescence signal of perfect duplexes in the biochip cells was calculated and then normalized to the value obtained for the commercially available Cy5-dUTP.

The formation of a full-sized PCR product depends not only on the chemical structure of the substituents, but also on the location of the substituent in the fluorophore molecule. Among the compounds of the general formula (TA), the most effective is the preparation with a sulfo group tightly bound to the dye indolenine fragment and farthest from the pyrimidine base of the nucleoside (TA4) - 6.75, while the effectiveness of the compound (TA3) with the sulfonate-butyl substituent is 5.25. When the sulfo group approaches the pyrimidine base of the triphosphate, the efficiency of the conjugate of the general formula (TA) decreases: (TA1) - 4.25, (TA5) - 3.9. It was also shown that the number of sulfo groups in the structure of the dye strongly affects the efficiency of the conjugate - regardless of the location of the carboxyl group. The fluorescent signals obtained for nucleotide (TA2) and commercially available Cy5-dUTP, were lower and approximately equal. In the series of compounds of the general formula TB, the drugs with the sulfonate-butyl substituent are most effective: (TB1) - 7.42, (TB2) - 7.11, while the compound (TB3) with the sulfo group tightly bound to the indolenin fragment of the dye and the most distant from the pyrimidine base of the nucleoside was less effective - 5.17.

Also, the efficiency of incorporation of modified nucleotides (TA) and (TB) under PCR conditions is presented using the PF-biochip Trombo test system to determine the genetic predisposition to thrombosis. Using fluorescently labeled nucleotides (TA) and (TB), the analysis of gene polymorphism of the fibrinolysis system (FGB, F7, GPIII a , PAI-1, MTHFR) was performed (Fig. 11). The procedure involves amplification of the sequence of selected genes, followed by hybridization of the labeled fragments with oligonucleotides immobilized in helium cells of the biochip.

The degree of inhibition of enzymatic activity was determined by performing symmetric PCR using a standard protocol in which the primers used were used in equal amounts. To determine the formation and size of the PCR products of the FGB, F7, GPIIIa, PAI-1, MTHFR genes, agarose gel electrophoresis was used. As can be seen from FIG. 12, FGB gene PCR products are located between the 242 bp and 100 bp bands of the pUC19 / MspI marker. Amplification using only native nucleotides (lane 9) and in the presence of modified substrates (lanes 1-8) led to the formation of a full-sized product. Electrophoregrams of the PCR products developed using ethidium bromide and upon recording the dye fluorescence signal were the same for the F7, GPIIIa, PAI-1 and MTHFR genes.

Hybridization on a biological microchip was carried out after two-round multiplex PCR with the formation of single-stranded fluorescently-labeled DNA fragments. Hybridization results are shown in FIG. 11.2 upper cells (perfect match) and two lower ones (mismatch) are duplicated. The fluorescent signal of the duplicated cells was averaged and normalized.

The efficiency of incorporation of modified nucleotides into PCR was determined by measuring the fluorescence of perfect duplexes for each analyzed oligonucleotide sample. The data obtained are corrected for the background signal and normalized to the value of Cy5-dUTP (Fig. 13). It was found that the efficiency of embedding modified nucleotides in PCR is similar in both test systems (“TB-biochip” and “PF-biochip Trombo”). The fluorescent signal is higher for the nucleotide (TA3), which contains in its structure a cyanine dye with a sulfo group remote from the pyrimidine base. However, in the case of the PF-Biochip Trombo test system, the most effective were the compounds of the general formula (TA), which were about 12 times more efficient than the signal obtained for Cy5-dUTP.

The use of fluorescently labeled nucleotides of the general formula (I) in PCR allows one to obtain a fluorescent signal 7 and 12 times higher than the commercially available product Cy5-dUTP in determining mutations of Mycobacterium tuberculosis and for detecting a genetic predisposition to thrombosis using the TB test systems -biochip ”and“ PF-biochip Trombo ”. Based on the foregoing, it follows that the inventive fluorescently-labeled deoxyuridine triphosphates of the general formula (I) (TA1-TA5, TB1-TB3) can be successfully used for DNA labeling during PCR.

Examples of compounds and their use

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

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 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 so that the temperature of the reaction mixture 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. Stirred for an additional 1 hour. Then the mixture was heated to boiling and concentrated hydrochloric acid (200 ml) was added dropwise, and a precipitate formed. 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 1) is obtained in a yield of 42 g (74%).

Preparation of 6-methyl-7-oxo-octanoic acid

Example 2. Obtaining 1-morpholinocyclohexene (compound 2)

A mixture of morpholine (100.0 ml, 1.15 mol), cyclohexanone (100.0 ml, 0.96 mol), toluene (190 ml) and n-toluenesulfonic acid (1.0 g) is boiled in a round bottom flask equipped with a reflux condenser and a Dean-Stark nozzle until the water separation ceases ( ~ 6 hours). Then the toluene is removed, and the residue is fractionated in vacuo, so Kip. 114 ° C (8 Torr). 111 g (69%) of 1-morpholinocyclohexene (compound 2) are obtained.

1 H NMR (CDCl 3, δ, ppm, J / Hz): 1.55 [m, 2H, C H2 (4)], 1.67 [m, 2H, C H ₂ (3)], 2.05 [m, 4H, C H ₂ (5), C H ₂ (6)], 2.77 (t, 4H, C H ₂ (2 '), C H ₂ (6'), J = 5), 3.72 (t, 4H, C H ₂ (3 '), CH ₂ (5'), J = 5), 4.66 (t, 1H, C H (2), J = 4)

Example 3. Obtaining 2-acetylcyclohexanone (compound 3)

Method A. Anhydrous chloroform (475 ml), 1-morpholinocyclohexene (compound 2) (63.0 g, 0.38 mol) and triethylamine (56.7 ml, distilled over sodium) are placed in a three-necked flask equipped with a reflux condenser, a mechanical stirrer, a thermometer and a dropping funnel. 0.41 mol). A solution of acetyl chloride (27.7 ml, 0.39 mol) in anhydrous chloroform (140 ml) was added dropwise to the reaction mixture cooled to 0 ° C so that the temperature of the mixture did not rise above 0 ° C. Additionally stirred under cooling on ice for 1 h, then the reaction mass is kept for 12 h at 20 ° C. Next, a solution of concentrated hydrochloric acid (50.4 ml) in water (126 ml) is added to the reaction mass and boiled for 5 hours with continuous stirring. After cooling, the chloroform layer is separated, washed with 200 ml portions of water (up to pH 5-6), saturated sodium chloride solution and dried with sodium sulfate. Chloroform is removed and the residue is fractionated in vacuo, boiling point. 102-104 ° C (10 Torr). 29.0 g (55%) of 2-acetylcyclohexanone (compound 3) are obtained.

NMR ¹ H (CDCl ₃ , δ, ppm): 1.67 [m, 4H, С Н ₂ (4), С Н ₂ (5)], 2.10 (s, 3H, COC H ₃ ), 2.31 [m , 4H, C H ₂ (3), C H ₂ (6)], 15.90 [s, 1H, C H (2)]

Method B. To a mixture of cyclohexanone (5.2 ml, 0.05 mol) and acetic anhydride (9.5 ml, 0.1 mol) cooled to 0 ° C, the BF ₃ ⋅ 2AcOH complex (13.9 ml, 0.1 mol) was introduced in one step and stirred for 24 hours at ~ 20 ° C. Then the reaction mass was added to a solution of CH ₃ COONa⋅3H ₂ O (27.2 g, 0.2 mol) in water (150 ml) and boiled for 3 h with vigorous stirring. After cooling, it was extracted with Et ₂ O (3 * 80 ml). The ether extracts were combined, washed with a saturated solution of NaHCO ₃ until gas ceased to stand out, and dried with MgSO ₄ . The ether was removed, and the residue was fractionated in vacuo under a stream of nitrogen, b.p. 105-108 ° C (17 Torr). 3.8 g (55%) of 2-acetylcyclohexanone (compound 3) was obtained.

NMR ¹ H (CDCl ₃ , δ, ppm): 1.67 [m, 4H, С Н ₂ (4), С Н ₂ (5)], 2.10 (s, 3H, COC H ₃ ), 2.31 [m , 4H, C H ₂ (3), C H ₂ (6)], 15.90 [s, 1H, C H (2)]

Example 4. Obtaining 2-acetyl-2-methylcyclohexanone (compound 4)

Methyl alcohol (83 ml), 2-acetylcyclohexanone (compound 3) (29.0 g, 0.21 mol) and methyl iodide (19.3 ml, 0.31 mol) are placed in a three-necked round bottom flask equipped with a reflux condenser, a mechanical stirrer and a thermometer while cooling on ice. . With stirring and cooling to 0 ° C, a solution of sodium hydroxide (8.3 g, 0.21 mol) in water (41.4 ml) is added dropwise to the reaction mass. The mixture is stirred for 20 hours at 20 ° C, and then boiled for an additional 2 hours. Next, water (85 ml) is added to the cooled reaction mass and extracted with chloroform (2 * 85 ml). The chloroform extracts are combined, washed with 2M sodium hydroxide (200 ml), saturated sodium chloride solution (100 ml) and dried with MgSO ₄ . The solvent is removed, and the residue is fractionated in vacuo in a stream of nitrogen, so Kip. 127-128 ° C (32 Torr). 24 g (75%) of 2-acetyl-2-methylcyclohexanone (compound 4) are obtained.

¹ H NMR (CDCl ₃ , δ, ppm): 1.23 (s, 3H, C (2) C H ₃ ), 1.41-1.48 [m, 1H, C H (4)], 1.61-1.69 [m , 3H, С Н (4), С Н ₂ (5)], 1.93-1.99 [m, 1Н, С Н (3)], 2.09 (s, 3H, COC H ₃ ), 2.25-2.34 [m, 1H , C H (3)], 2.42-2.48 [m, 2H, C H ₂ (6)]

Example 5. Obtaining methyl ester of 6-methyl-7-oxo-octanoic acid (compound 5)

A mixture of anhydrous MeOH (52.4 ml), Li (0.5 g, 0.071 mol) and 2-acetyl-2-methylcyclohexanone (compound 4) (10.0 g, 0.065 mol) was boiled for 1 h with stirring, and then kept at ~ 20 ° for 12 h C. Next, the reaction mass was acidified with concentrated H ₂ SO ₄ to pH 4-5. Water (55 ml) was added to the suspension and extracted with Et ₂ O until the aqueous phase decolored. The ether extracts were combined, washed with saturated NaCl and dried with MgSO ₄ . The solvent was removed, and the residue was fractionated in vacuo, boiling point. 139-143 ° C (20 Torr). Received 6.2 g (52%) of methyl ester of 6-methyl-7-oxo-octanoic acid (compound 5).

¹ H NMR spectrum (CDCl ₃ , δ, ppm, J / Hz): 1.06 (d, 3H, C (6) C H ₃ , J = 7), 1.23-1.35 [m, 3H, C H ₂ (5), C H (4)], 1.56-1.69 [m, 3 H, C H ₂ (3), C H (4)], 2.11 [s, 3H, C H ₃ (8)], 2.28 ( t, 2 N, С Н ₂ (2), J = 7.5), 2.49 [m, 1 Н, С Н (6)], 3.63 (s, 3H, COOC H ₃ )

Example 6. Obtaining 6-methyl-7-oxo-octanoic acid (compound 6)

A mixture of 12% HCl (37 ml) and 6-methyl-7-oxo-octanoic acid methyl ester (compound 5) (9.1 g, 0.049 mol) was boiled for 6 hours with stirring. The reaction mass was extracted with ether (50 ml). The solvent was removed, and the residue was fractionated in vacuo, boiling point. 122 ° C (0.1-0.01 Torr). Received 5.9 g (75%) of compound 6.

¹ H NMR (CDCl ₃ , δ, ppm, J / Hz): 1.06 (d, 3H, C (6) C H ₃ , J = 7), 1.25-1.39 [m, 3H, C H ₂ ( 5), С Н (4)], 1.58-1.69 [m, 3H, С Н ₂ (3), С Н (4)], 2.12 [s, 3H, С Н ₃ (8)], 2.33 (t, 2H, C H ₂ (2), J = 7.5), 2.50 [m, 1H, C H (6)]

4- (2-carboxyethyl) phenylhydrazine hydrochloride

Example 7. Obtaining 2-chloro-3- (4-nitrophenyl) propionitrile (compound 12)

A mixture of 4-nitroaniline (11.5 g, 0.083 mol), water (60 ml) and concentrated HCl (62 ml) is heated to completely dissolve the released 4-nitroaniline hydrochloride. Then the reaction mass is cooled until crystallization begins, finely crushed ice (50 g) is added, and a solution of NaNO ₂ (5.9 g, 0.083 mol) in water (13.8 ml) is added dropwise to the resulting suspension with vigorous stirring. The progress of the reaction is monitored by iodine starch paper. To the resulting solution, with stirring, add a solution of acrylonitrile (4.4 g, 0.083 mol) in acetone (37.5 ml), crystalline CuSO ₄ ⋅ 5H ₂ O (2.5 g, 0.01 mol) and the mixture was incubated for 24 h at ~ 20 ° C. Then, the precipitate formed is filtered off, washed with water, transferred to a flask with water (250 ml) and the impurities are removed by steam distillation. After collecting 500 ml of condensate, distillation is stopped. The bottom residue is cooled on ice and the aqueous layer is removed by reverse filtration. The solid residue was dissolved in methanol (100 ml), treated with activated carbon (1 g), the solution was filtered hot and water (300 ml) was added. The precipitate formed is filtered off and dried at 80 ° C. Obtain 14.3 g (82%) of nitrile (compound 12), so pl. 110-111 ° C.

¹ N (DMSO-d ₆ , δ, ppm, J / Hz): 3.55 (m, 2H, C H ₂ CH (Cl) CN), 5.64 (t, 1H, CH ₂ C H (Cl) CN , J = 7.0), 7.65-8.26 (d, 4H, ArH, J = 9.0)

Example 8. Obtaining 3- (4-nitrophenyl) -acrylonitrile (compound 13)

A mixture of 2-chloro-3- (4-nitrophenyl) propionitrile (compound 12) (10.5 g, 0.05 mol), sodium acetate (15.0 g, 0.18 mol), water (40 ml) and ethyl alcohol (90 ml) is boiled in for 4 hours. Then water (150 ml) was added to the reaction mass and kept at + 5 ° C until complete crystallization. The resulting precipitate was collected by filtration, dissolved in ethanol (200 ml), treated with activated carbon (1.5 g), the solution was filtered hot. Water (400 ml) was added to the filtrate, incubated for 24 hours at + 5 ° C and the precipitate formed was filtered off. Obtain 8.2 g of technical compound 13. After additional recrystallization from alcohol (200 ml), 4.1 g (47%) of 3- (4-nitrophenyl) acrylonitrile (compound 13) are obtained in the form of yellow needles, mp. 201 ° C (from ethanol).

¹ H NMR (DMSO-d ₆ , δ, ppm, J / Hz): 6.72 (d, 1H, CHC H CN, J = 16.5), 7.81 (d, 1H, C H CHCN, J = 16.5) 7.79-8.29 (d, 4H, ArH, J = 9.0)

Example 9. Obtaining 3- (4-nitrophenyl) -2-propenoic acid (compound 14)

A mixture of 3- (4-nitrophenyl) acrylonitrile (compound 13) (17.4 g, 0.1 mol) and concentrated phosphoric acid (350 ml) was heated for 2 hours at 150 ° C. After cooling to 20 ° C, the mixture was transferred to ice water (4 L). The resulting precipitate is filtered off. Obtain 18.3 g of the crude product, which is dissolved in 1 l of a 0.12M NaOH solution and treated with charcoal (3.5 g). The resulting solution was filtered off and concentrated HCl was added dropwise to the filtrate to a pH of ~ 2. The precipitate formed is filtered off, washed with water and dried at 90 ° C. Obtain 17.7 g (92%) of acid (compound 14) in the form of yellow needles, so pl. > 250 ° C.

¹ H NMR (DMSO-d ₆ , δ, ppm, J / Hz): 6.74 (d, 1H, CHC H COOH, J = 16.5), 7.69 (d, 1H, C H CHCOOH, J = 16.5) 7.96-8.25 (d, 4H, ArH, J = 9)

Example 10. Obtaining hydrochloride of 3- (4-aminophenyl) -propionic acid (compound 15)

A mixture of 5% Pd / C catalyst (400 mg), acetic acid (80 ml) and 3- (4-nitrophenyl) -2-propenoic acid (compound 14) (3.9 g, 20 mmol) was hydrogenated for 2.5 h with vigorous stirring in a stream hydrogen at ~ 1 atm and ~ 20 ° C. The reaction mass is filtered off and the filtrate is evaporated. The oily residue is dissolved in a mixture of 5M HCl solution (7 ml) and water (30 ml), treated with activated charcoal when heated. Obtain 3.9 g (96%) of the hydrochloride (compound 15), so pl. 186-187 ° C.

¹ H NMR (DMSO-d ₆ , δ, ppm, J / Hz): 2.53 (t, 2H, CH ₂ C H ₂ COOH, J = 7.5), 2.83 (t, 2H, C H ₂ CH ₂ COOH, J = 7.5), 7.28-7.35 (d, 4H, ArH, J = 8.5), 10.31 (s, 2H, N H ₂ )

Example 11. Obtaining 4- (2-carboxyethyl) phenylhydrazine hydrochloride (compound 16)

In a three-necked flask equipped with a mechanical stirrer and a thermometer, 3- (4-aminophenyl) propionic acid hydrochloride (compound 15) (2.0 g, 10 mmol), concentrated hydrochloric acid (35 ml) were placed and, while cooling, a solution was added dropwise under a liquid layer sodium nitrite (0.69 g, 10 mmol) in water (0.9 ml) at such a rate that the temperature of the reaction mass does not rise above -5 ° C. Then within 30 minutes the temperature was raised to 0 ° C. The mixture is again cooled to -5 ° C and a solution of SnCl ₂ ⋅ 2H ₂ O (10.0 g, 44 mmol) in concentrated hydrochloric acid (15 ml) is added at a rate such that the temperature of the reaction mass does not rise above - 5 ° C. The mixture is further stirred for 30 minutes at -5 ° C and incubated for -24 hours at + 5 ° C. The precipitated crystals are filtered off and washed with concentrated hydrochloric acid (6 ml) cooled to -18 ° C. Obtain 1.6 g (75%) of the hydrochloride (compound 16).

¹ H NMR (DMSO-d ₆ , δ, ppm, J / Hz): 2.47 (t, 2H, CH ₂ C H ₂ COOH, J = 7.5), 2.73 (t, 2H, C H ₂ CH ₂ COOH, J = 7.5), 6.91-7.13 (d, 4H, ArH, J = 8.5), 10.27 (s, 3H, N H N H ₂ )

Synthesis of Indolenins

Example 12. Obtaining 3- (4-carboxybutyl) -2,3-dimethylindolenine (compound 9)

A mixture of 6-methyl-7-oxo-octanoic acid (compound 6) (723 mg, 4.2 mmol), phenylhydrazine (454 mg, 4.2 mmol) and glacial acetic acid (4.2 ml) was heated for 3.5 hours at 118 ° C. The solvent is removed, the oily residue is dissolved in chloroform. The chloroform solution was washed with water, saturated sodium chloride solution, dried with MgSO ₄ . The solvent is removed, and the residue is recrystallized from a mixture of ethyl acetate - hexane, 0.6: 2. Obtain 0.8 g (78%) of indolenin (compound 9) in the form of a yellow-orange powder. Mass spectrum (MALDI) (C ₁₅ H ₁₉ NO ₂ ): found m / z 246.3, calculated m / z 245.32.

¹ H NMR (CDCl ₃ , δ, ppm): 0.56-0.81 (m, 2H, CH ₂ C H ₂ CH ₂ CH ₂ COOH), 1.28 [s, 3H, CH ₃ (3)], 1.43- 1.51 (m, 2H, C H ₂ CH ₂ CH ₂ CH ₂ COOH), 1.76-1.92 (m, 2H, CH ₂ CH ₂ C H ₂ CH ₂ COOH), 2.13-2.21 (m, 2H, CH ₂ CH ₂ CH ₂ C H ₂ COOH), 2.24 [s, 3H, CH ₃ (2)], 7.18-7.54 (m, 4H, Ar H)

Example 13. Obtaining the potassium salt of 3- (4-carboxybutyl) -2,3-dimethyl-5-sulfoindolenine (compound 7)

A mixture of phenylhydrazine-i-sulfonic acid (compound 1) (1.8 g, 9.7 mmol), 6-methyl-7-oxo-octanoic acid (compound 6) (2.0 g, 11.6 mmol) and glacial acetic acid (15 ml) is boiled under stirring for 8 hours, then additional acetic acid is added and treated with activated carbon. The solvent was removed and the residue was dissolved in methanol (10 ml). To the resulting solution was added dropwise a solution of potassium hydroxide (600 mg, 10.6 mmol) in methanol (7 ml), isopropyl alcohol (15 ml) and diethyl ether (2 ml). The resulting precipitate was filtered and dried in a vacuum desiccator over P ₂ O _5. Obtain 3.5 g (83%) of indolenin (compound 7). λ _max 258 nm. Mass spectrum (MALDI) (C ₁₅ H ₁₉ NO ₅ S): found m / z 327.2, calculated m / z 325.38.

¹ H NMR (D ₂ O, δ, ppm, J / Hz): 0.45-0.70 (m, 2H, CH ₂ C H ₂ CH ₂ CH ₂ COOH), 1.31 [s, 3H, CH ₃ (3 )], 1.36 (m, 2H, C H ₂ CH ₂ CH ₂ CH ₂ COOH), 1.88-2.14 (m, 4H, CH ₂ CH ₂ C H ₂ C H ₂ COOH), 7.53 (d, 1H, Ar H , J = 9), 7.80 (m, 2H, Ar H )

Example 14. Obtaining 5- (2-carboxyethyl) -2,3,3-trimethylindolenine (compound 17)

A mixture of 4- (2-carboxyethyl) phenylhydrazine hydrochloride (compound 16) (216 mg, 1 mmol), anhydrous potassium acetate (196 mg, 2.0 mmol), 3-methyl-2-butanone (147 μl, 1.4 mmol), acetic acids (1 ml) are heated for 1 h at 118 ° C. Then the solvents are removed, the residue is dissolved in chloroform, washed with water, a saturated solution of sodium chloride and dried with sodium sulfate. Chloroform was removed and the oily residue was recrystallized from ethyl acetate: hexane 1:10. The precipitate was collected by filtration and dried in a vacuum desiccator over P ₂ O ₅ and KOH. 118 mg (55%) of indolenine (compound 17) are obtained. λ _max 262 nm. Mass spectrum (MALDI) (C ₁₄ H ₁₇ NO ₂ ): found m / z 232.3, calculated m / z 231.29.

¹ H NMR (CDCl ₃ , δ, ppm, J / Hz): 1.27 (s, 6H, 2C (3) H ₃ ); 2.27 (s, 3H, C (2) H ₃ ), 2.68 (t, 2H, CH ₂ C H ₂ COOH, J = 8), 3.00 (t, 2H, C H ₂ CH ₂ COOH, J = 8), 7.12-7.46 (d, 3H, Ar, J = 8.5)

Example 15. Obtaining a potassium salt of 2,3,3-trimethyl-5-sulfoindolenine (compound 20)

A mixture of phenylhydrazine-n-sulfonic acid (compound 1) (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). A solution of potassium hydroxide (1.4 g, 0.024 mol) in methanol (16 ml) was added dropwise to the resulting solution. 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 20) 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.

NMR ¹ H (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 )

N-alkylation of indolenins

Example 16. Obtaining 3- (4-carboxybutyl) -2,3-dimethyl-N-ethylindoleninium iodide (compound 10)

A mixture of 2,3-dimethyl-3- (4-carboxybutyl) indolenine (compound 9) (49 mg, 0.2 mmol), ethyl iodide (1 ml) and acetonitrile (0.4 ml) was heated at 70 ° C for 20 hours. At the end of heating, the solvent was removed in vacuo, and the residue was washed with diethyl ether and dried in a vacuum desiccator over P ₂ O ₅ . 76 mg (95%) of 3- (4-carboxybutyl) -2,3-dimethyl-N-ethylindoleninium iodide (compound 10) is obtained. λ _max 279 nm. Mass spectrum (MALDI) (C ₁₇ H ₂₄ NO ₂ ⁺ ): found m / z 274.4, calculated m / z 274.38.

¹ H NMR (CDCl ₃ , δ, ppm, J / Hz): 0.52-0.78 (m, 2H, CH ₂ C H ₂ CH ₂ CH ₂ COOH), 1.25 (s, 3H, C (3) N _3), 1.38-1.49 (m, 2H, C H ₂ CH ₂ CH ₂ CH ₂ COOH), 1.62 (t, 3H, CH ₂ CH _3, J = 7.0), 1.70-1.81 (m, 2H, CH ₂ CH ₂ C H ₂ CH ₂ COOH), 2.18 (m, 2H, CH ₂ CH ₂ CH ₂ C H ₂ COOH), 2.28 (s, 3H, C (2) H ₃ ), 4.73 (m, 2H, C H ₂ CH ₃ ), 7.5-7.68 (m, 4H, Ar H )

Example 17. Obtaining 2,3-dimethyl-3- (4-carboxybutyl) -5-sulfo-N-ethylindoleninium (compound 8)

A mixture of the potassium salt of 3- (4-carboxybutyl) -2,3-dimethyl-5-sulfoindolenine (compound 7) (0.5 g, 1.4 mmol), ethyl iodide (550 μl, 6.9 mmol) and 1,2-dichlorobenzene (25 μl) heated for 50 hours at 72 ° C. At the end of heating, the solvent is decanted, the precipitate is washed with acetone, collected on a filter and dried in a vacuum desiccator over P ₂ O ₅ . Obtain 0.47 g (96%) of 3- (4-carboxybutyl) -2,3-dimethyl-5-sulfo-N-ethylindoleninium (compound 8). λ _max 266 nm. Mass spectrum (MALDI) (C ₁₇ H ₂₃ NO ₅ S): found m / z 354.2, calculated m / z 353.43.

¹ H NMR (D ₂ O, δ, ppm, J / Hz): 0.58-0.72 (m, 2H, CH ₂ C H ₂ CH ₂ CH ₂ COOH), 1.49 (t, 3H, CH ₂ CH ₃ , J = 7), 1.56 (s, 3H, C (3) H ₃ ), 1.61 (m, 2H, C H ₂ CH ₂ CH ₂ CH ₂ COOH), 2.14 (br.m, 4H, CH ₂ CH ₂ C H ₂ C H ₂ COOH), 4.5 (m, 2H, C H ₂ CH ₃ ), 7.9 (d, 1H, ArH, J = 9), 8.06 (m, 2H, ArH)

Example 18. Obtaining 2,3,3-trimethyl-N-ethylindoleninium iodide (compound 23)

A mixture of 2,3,3-trimethylindolenine (1.4 g, 8.5 mmol), ethyl iodide (5.5 ml) and acetonitrile (5 ml) was boiled for 18 hours under stirring with a stream of nitrogen. At the end of heating, anhydrous diethyl ether (30 ml) was added to the reaction mixture cooled to room temperature. The precipitated crystals are collected on a filter and recrystallized from isopropanol (5 ml). Obtain 2.5 g (92%) of 2,3,3-trimethyl-N-ethylindoleninium iodide (compound 23). λ _max 278 nm. Mass spectrum (MALDI) (C ₁₃ H ₁₈ N ⁺ ): found m / z 189.3, calculated m / z 188.29.

¹ H NMR (CDCl ₃ , δ, ppm): 1.64 (br s, 9H, CH ₂ C H ₃ , 2C (3) H ₃ ), 3.14 (s, 3H, C (2) H ₃ ) 4.75 (m, 2H, C H ₂ CH ₃ ), 7.56-7.72 (m, 4H, ArH)

Example 19. Obtaining 2,3,3-trimethyl-5-sulfo-N-ethylindoleninium (compound 21)

A mixture of potassium salt of 2,3,3-trimethyl-5-sulfoindolenine (compound 20) (2.4 g, 10 mmol), triethylamine (1.7 ml, 12 mmol), n-toluenesulfonic acid ethyl ester (5.0 g, 25 mmol) and butyronitrile ( 10 ml) is heated for 20 hours at 118 ° C. At the end of heating, acetone (30 ml) was added. The crystals formed are filtered off and dried in a vacuum desiccator over P ₂ O ₅ . Obtain 2.32 g (86%) of indoleninium salt (compound 21), so pl. 240-242 ° C. λ _max 228 nm. Mass spectrum (MALDI) (C ₁₃ H ₁₇ NO ₃ S): found m / z 268.4, calculated m / z 267.35.

¹ H NMR (D ₂ O, δ, ppm, J / Hz): 1.43 (br s, 9H, CH ₂ C H ₃ , 2C (3) H ₃ ), 4.36 (m, 2H, C H ₂ CH ₃ ), 7.63 (d, 1H, ArH, J = 8.5), 7.75 (d, 1H, ArH, J = 8.5), 7.97 (s, 1H, ArH)

Example 20. Obtaining 5- (2-carboxyethyl) -2,3,3-trimethyl-N-ethylindoleninium iodide (compound 18)

A mixture of 5- (2-carboxyethyl) -2,3,3-trimethylindolenine (compound 17) (230 mg, 1 mmol), ethyl iodide (500 μl, 5.5 mmol), acetonitrile (2.2 ml) was heated at 70 ° C for 20 hours. Then it was cooled to room temperature, absolute diethyl ether (2 ml) was added and incubated for ~ 24 h at -18 ° C. The obtained crystals are filtered off and dried in a vacuum desiccator over P ₂ O ₅ and KOH. 377 mg (97%) of indoleninium iodide (compound 18) is obtained. λ _max 284 nm. Mass spectrum (MALDI) (C ₁₆ H ₂₁ NO ₂ ): found m / z 259.8, calculated m / z 259.34.

¹ H NMR (DMSO-d ₆ , δ, ppm, J / Hz): 1.44 (t, 3H, CH ₂ C H ₃ , J = 7.0), 1.52 (s, 6H, 2C (3) H ₃ ), 2.62 (t, 2H, CH ₂ C H ₂ COOH, J = 7.5), 2.80 (s, 3H, C (2) H ₃ ), 2.96 (t, 2H, C H ₂ CH ₂ COOH, J = 7.5 ), 4.47 (q, 2H, C H ₂ CH ₃ , J = 7.0), 7.47-7.86 (m, 3H, ArH)

N-sulfoalkylation of indolenins

Example 21. Obtaining 5- (2-carboxyethyl) -2,3,3-trimethyl-N- (4-sulfonateobutyl) indoleninium (compound 19)

A mixture of 5- (2-carboxyethyl) -2,3,3-trimethylindolenine (compound 17) (230 mg, 1 mmol), 1,4-butanesultone (216 mg 1.6 mmol) and butyronitrile (2.2 ml) was heated for 22 hours at 118 ° C. Cooled to room temperature, the reaction mass is triturated with absolute diethyl ether. The precipitate formed is filtered off and dried in a vacuum desiccator over P ₂ O ₅ and KOH. 300 mg (86%) of indoleninium (compound 19) are obtained. λ _max 284 nm. Mass spectrum (MALDI) (C ₁₈ H ₂₅ NO ₅ S): found m / z 368.2, calculated m / z 367.46.

¹ H NMR (DMSO-d6, δ, ppm, J / Hz): 1.51 (s, 6H, 2C (3) H ₃ ), 1.73 (m, 2H, CH ₂ C H ₂ CH ₂ CH ₂ SO ₃ ), 1.96 (m, 2H, CH ₂ CH ₂ C H ₂ CH ₂ SO ₃ ), 2.61 (t, 2H, CH ₂ C H ₂ COOH, J = 7.5), 2.81 (s, 3H, C (2) H ₃ ), 2.85 (m, 2H, CH ₂ CH ₂ CH ₂ C H ₂ SO ₃ ), 2.95 (t, 2H, C H ₂ CH ₂ COOH, J = 7.5), 4.46 (m, 2H, C H ₂ CH ₂ CH ₂ CH ₂ SO _3), 7.47-7.93 (m, 3H, ArH)

Example 22. Obtaining 2,3,3-trimethyl-N- (4-sulfonateobutyl) indoleninium (compound 22)

A mixture of 2,3,3-trimethylindolenine (414 mg, 2.6 mmol), 1,4-butanesultone (708 mg, 5.2 mmol) and 1,2-dichlorobenzene (4.2 ml) was heated at 118 ° C for 25 hours. The solvent is decanted and the residue is triturated with diethyl ether. The precipitate formed is collected on a filter, washed with diethyl ether and dried in a vacuum desiccator over P ₂ O ₅ . Obtain 0.75 g (97%) of indoleninium (compound 22) in the form of a dark cherry powder. λ _max 278 nm. Mass spectrum (MALDI) (C ₁₅ H ₂₁ NO ₃ S): found m / z 296.0, calculated m / z 295.40.

¹ H NMR (D ₂ O, δ, ppm, J / Hz): 1.44 (s, 6H, 2C (3) H ₃ ), 1.77 (m, 2H, CH ₂ CH ₂ C H ₂ CH ₂ SO ₃ ), 2.02 (m, 2H, CH ₂ C H ₂ CH ₂ CH ₂ SO ₃ ), 2.84 (t, 2H, CH ₂ CH ₂ CH ₂ C H ₂ SO ₃ , J = 7.5), 4.39 (t, 2H , C H ₂ CH ₂ CH ₂ SO ₂ CH _3, J = 7.5), 7.49-7.68 (m, 4H, ArH)

Example 23. Obtaining 3- (4-carboxybutyl) -2,3-dimethyl-N- (4-sulfonate-butyl) indoleninium (compound 11)

A mixture of 3- (4-carboxybutyl) -2,3-dimethylindolenine (compound 9) (638 mg, 2.6 mmol), 1,4-butanesultone (708 mg, 5.2 mmol) and 1,2-dichlorobenzene (4.2 ml) is heated 25 h at 118 ° C. The solvent is decanted and the oily residue is triturated with diethyl ether. The resulting precipitate was collected by filtration, washed with diethyl ether and dried in a vacuum desiccator over P ₂ O _5. 0.98 g (99%) of indoleninium (compound 11) is obtained in the form of a dark cherry powder. λ _max 280 nm. Mass spectrum (MALDI) (C ₁₉ H ₂₇ NO ₅ S): found m / z 382.0, calculated m / z 381.49.

¹ H NMR (D ₂ O, δ, ppm, J / Hz): 0.38-0.68 (m, 2H, C H ₂ CH ₂ CH ₂ CH ₂ COOH), 1.23-1.35 (m, 2H, C H ₂ CH ₂ CH ₂ CH ₂ COOH), 1.43 (s, 3H, C (3) H ₃ ), 1.71-1.81 (m, 2H, CH ₂ C H ₂ CH ₂ CH ₂ SO ₃ ), 1.92-2.2 (br ppm, 6H, CH ₂ CH ₂ C H ₂ CH ₂ COOH, CH ₂ CH ₂ C H ₂ CH ₂ SO ₃ ), 2.84 (t, 2H, CH ₂ CH ₂ CH ₂ C H ₂ SO ₃ , J = 7.5), 4.41 (t, 2H, C H ₂ CH ₂ CH ₂ CH ₂ SO ₃ , J = 7.5), 7.18-7.7 (br.m, 4H, ArH)

Synthesis of diphenylimine hydrochloride malondialdehyde

Example 24. Obtaining bis- (diethylacetal) malondialdehyde (compound 24)

Ethyl orthoformate (50.0 ml, 0.3 mol) and boron trifluoride etherate (0.5 ml) are added to a three-necked flask equipped with a reflux condenser, a mechanical stirrer, a thermometer and a dropping funnel, and vinyl ethyl ether (27 ml, 0.28 mol) is added dropwise with such speed so that the temperature of the mixture does not rise above 38 ° C. Additionally stirred for 1 h at 20 ° C. Then anhydrous potassium carbonate (3 g, 0.02 mol) was added to the reaction mass and stirred for 2 hours at 20 ° C. The precipitate formed is filtered off, and the filtrate is fractionated in vacuo in a stream of nitrogen, b.p. 101-102 ° C (12 Torr). Bis (diethylacetal) malondialdehyde (compound 24) is obtained in a yield of 43 g (69%). Mass spectrum (MALDI) (C ₁₁ H ₂₄ O ₄ ): found m / z 221.5, calculated m / z 220.31.

¹ H NMR (CDCl ₃ , δ, ppm, J / Hz): 1.20 (t, 12H, OCH ₂ C H ₃ , J = 7); 1.96 (t, 2H, CH ₂ , J = 6); 3.46-3.71 (m, 8H, OS H ₂ CH ₃ ); 4.62 (t, 2H, 2CH, J = 6)

Example 25. Obtaining dianyl hydrochloride of malondialdehyde (compound 25)

A mixture of aniline hydrochloride (4. 7 g, 36.4 mmol) and methanol (6 ml) was heated until a homogeneous solution was formed, to which diethyl acetal malondialdehyde (compound 24) (4.0 g, 18.2 mmol) was added dropwise. Then the suspension is boiled for 10 min, cooled to ~ 20 ° C, water (500 ml) is added and incubated for 24 hours at + 5 ° C. The precipitate formed is filtered off, washed with ice water and dried in a vacuum desiccator over P ₂ O ₅ and KOH. Obtain 4.2 g (89%) of the hydrochloride (compound 25) in the form of small crystals of orange-yellow color. Mass spectrum (MALDI) (C ₁₅ H ₁₅ N ₂ ⁺ ): found m / z 224.5, calculated m / z 223.29

¹ H NMR (DMSO-d ₆ , δ, ppm, J / Hz): 6.48 (t, 2H, β-CH, J = 11.5); 7.4 (s, 10H, ArH); 8.9 (t, 2H, α, α'-CH, J = 12.0); 12.49 (d, 2H, NH, J = 13.0)

Dye synthesis

Example 26. Obtaining 3- (4-carboxybutyl) -3,3 ', 3'-trimethyl-N- (4-sulfonate-butyl) -N’-ethylindodicarbocyanine dye (compound A1)

A mixture of 2,3,3-trimethyl-N-ethylindoleninium iodide (compound 23) (63 mg, 0.2 mmol), malonic dialdehyde dianyl hydrochloride (compound 25) (52 mg, 0.2 mmol), acetic anhydride (700 μl) and acetic acid (300 μl) is heated for 2 hours at 118 ° C. At the end of heating, the solvent was removed in vacuo. The residue was dissolved in acetic anhydride (1.7 ml) and a solution of 3- (4-carboxybutyl) -2,3-dimethyl-N- (4-sulfonate-butyl) indoleninium (compound 11) (80 mg, 0.21 mmol) in a mixture of diisopropylethylamine ( 200 μl, 2.1 mmol) and acetic anhydride (800 μl). The reaction mass is heated for 3 hours at 60 ° 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) is added and the reaction mass is neutralized with a NaHCO ₃ solution to pH 7. The reaction products are isolated by reverse phase chromatography on an RP-18 column in a MeCN-0.05 M triethyl ammonium acetate buffer solution with gradient elution from 0 to 50% MeCN. 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 acetonitrile-water system. The solvent is removed in vacuo, the residue is dried in a vacuum desiccator over P ₂ O ₅ . Get 46 mg (38%) of compound A1, so pl. 175-176 ° C. Found: m / z 605.7 [M] ⁺ . C ₃₅ H ₄₄ N ₂ O ₅ S. Calculated: M = 604.80.

¹ H NMR (DMSO-d ₆ , δ, ppm, J / Hz): 0.43 (m, 1H, CH ₂ C H ₂ CH ₂ CH ₂ COOH), 0.78 (m, 1H, CH ₂ C H ₂ CH ₂ CH ₂ COOH), 1.24 (m, 3H, CH ₂ C H ₃ ), 1.29 (m, 2H, C H ₂ CH ₂ CH ₂ CH ₂ COOH), 1.64 (s, 9H, C (3) H ₃ , C (3 ', 3') H ₃ ), 1.73 (m, 4H, CH ₂ C H ₂ CH ₂ CH ₂ SO ₃ ), 1.89 (m, 2H, CH ₂ CH ₂ C H ₂ CH ₂ COOH), 2.18 (m, 1H, CH ₂ CH ₂ CH ₂ C H ₂ COOH), 2.41 (m, 3H, CH ₂ CH ₂ CH ₂ C H ₂ COOH, CH ₂ CH ₂ CH ₂ C H ₂ SO ₃ ), 4.12 ( m, 4H, C H ₂ CH ₃ , C H ₂ CH ₂ CH ₂ CH ₂ SO ₃ ), 6.26 (d, 1H, α'-CH, J = 13.5), 6.41 (d, 1H, α-CH, J = 13.5), 6.57 (t, 1H, γ-CH, J = 12.0), 7.21-7.61 (br.m, 8H, ArH), 8.32 (m, 2H, β, β'-CH)

Example 27. Obtaining the sodium salt of 3- (4-carboxybutyl) -3,3 ', 3'-trimethyl-N, N’-di (4-sulfonate-butyl) -indodicarbocyanine dye (compound A2).

A mixture of 2,3,3-trimethyl-N- (4-sulfonate-butyl) indoleninium (compound 22) (59 mg, 0.2 mmol), malonic dialdehyde dianyl hydrochloride (compound 25) (52 mg, 0.2 mmol), acetic anhydride (700 μl) and acetic acid (160 μl) are heated for 2 hours at 118 ° C. 3- (4-carboxybutyl) -2,3-dimethyl-N- (4-sulfonate-butyl) indoleninium (compound 11) (80 mg, 0.21 mmol), anhydrous potassium acetate (117 mg, 1.2 mmol) are added to the reaction mass; acetic anhydride (700 μl), acetic acid (350 μl) and heated for an additional 2 hours at 118 ° C. The reaction product is isolated analogously to compound A1. Obtain 71 mg (48%) of compound A2. Mass spectrum (MALDI) (C ₃₇ H ₄₇ N ₂ O ₈ S ^- ): found m / z 712.1, calculated m / z 711.91.

¹ H NMR (DMSO-d ₆ , δ, ppm, J / Hz): 0.45 (m, 1H, CH ₂ CH ₂ C H ₂ CH ₂ COOH), 0.77 (m, 1H, CH ₂ CH ₂ C H ₂ CH ₂ COOH), 1.32 (m, 2H, CH ₂ C H ₂ CH ₂ CH ₂ COOH), 1.65-1.8 (m, 17H, C (3) H ₃ , C (3 ', 3') H ₃ , CH ₂ C H ₂ C H ₂ CH ₂ SO ₃ ), 1.91 (m, 2H, C H ₂ CH ₂ CH ₂ CH ₂ COOH), 2.17 (m, 1H, CH ₂ CH ₂ CH ₂ CH ₂ C H ₂ COOH) 2.42 (m, 5H, CH ₂ CH ₂ CH ₂ C H ₂ COOH, CH ₂ CH ₂ CH ₂ C H ₂ SO ₃ ), 4.09 (m, 4H, C H ₂ CH ₂ CH ₂ CH ₂ SO ₃ ), 6.37 (m, 2H, α, α'-CH), 6.57 (t, 1H, γ-C H, J = 12.0), 7.21-7.6 (m, 8H, Ar H), 8.3 (m, 2H, β, β'-C H )

Example 28. Obtaining 3- (4-carboxybutyl) -3,3 ', 3'-trimethyl-N ’- (4-sulfonate-butyl) -N-ethylindodicarbocyanine dye (compound A3)

3- (4-Carboxybutyl) -3,3 ', 3'-trimethyl-N' - (4-sulfonate-butyl) -N-ethylindodicarbocyanine dye (compound A3) was obtained analogously to compound A1 in the following reagent ratios: (1 step) 3- (4-carboxybutyl) -2,3-dimethyl-N-ethylindoleninium iodide (compound 10) (80 mg, 0.2 mmol), malonic dialdehyde dianyl hydrochloride (compound 25) (73 mg, 0.28 mmol), acetic anhydride (3 ml) acetyl chloride (65 μl); (2 stage) 2,3,3-trimethyl-N- (4-sulfonate-butyl) indoleninium (compound 22) (149 mg, 0.5 mmol), acetic anhydride (3 ml), diisopropylethylamine (120 μl, 1.25 mmol) . 34 mg (28%) of compound A3 are obtained. Mass spectrum (MALDI) (C ₃₅ H ₄₄ N ₂ O ₅ S): found m / z 605.7, calculated m / z 604.80.

¹ H NMR (DMSO-d ₆ , δ, ppm, J / Hz): 0.48 (m, 1H, CH ₂ CH ₂ C H ₂ CH ₂ COOH), 0.79 (m, 1H, CH ₂ CH ₂ C H ₂ CH ₂ COOH), 1.2-1.35 (m, 5H, CH ₂ C H ₃ , CH ₂ C H ₂ CH ₂ CH ₂ COOH), 1.65-1.81 (m, 13H, C (3) H ₃ , C ( 3 ', 3') H ₃ , CH ₂ C H ₂ C H ₂ CH ₂ SO ₃ ), 2.02 (m, 2H, C H ₂ CH ₂ CH ₂ CH ₂ COOH), 2.17 (m, 1H, CH ₂ CH ₂ CH ₂ C H ₂ COOH), 2.34 (m, 3H, CH ₂ CH ₂ CH ₂ C H ₂ COOH, CH ₂ CH ₂ CH ₂ C H ₂ SO ₃ ), 4.12 (m, 4H, C H ₂ CH ₃ , C H ₂ CH ₂ CH ₂ CH ₂ SO ₃ ), 6.35 (m, 2H, α, α'-C H ), 6.58 (m, 1H, γ-C H ), 7.22-7.61 (m, 8H, Ar H ), 8.3 (m, 2H, β, β'-C H )

Example 29. Obtaining 3- (4-carboxybutyl) -3,3 ', 3'-trimethyl-5'-sulfo-N, N’-diethylindodicarbocyanine dye (compound A4).

3- (4-Carboxybutyl) -3,3 ', 3'-trimethyl-5'-sulfo-N, N'-diethylindodicarbocyanine dye (compound A4) is obtained analogously to compound A1 with the following reagent ratios: (1 step) 3- ( 4-carboxybutyl) -2,3-dimethyl-N-ethylindoleninium iodide (compound 10) (80 mg, 0.2 mmol), malonic dialdehyde dianyl hydrochloride (compound 25) (73 mg, 0.28 mmol), acetic anhydride (3 ml), acetyl chloride (65 μl); (2 stage) 2,3,3-trimethyl-5-sulfo-N-ethylindoleninium (compound 21) (134 mg, 0.5 mmol), acetic anhydride (3 ml), diisopropylethylamine (120 μl, 1.25 mmol). Obtain 31 mg (27%) of compound A4. Mass spectrum (MALDI) (C ₃₃ H ₄₀ N ₂ O ₅ S): found m / z 577.0, calculated m / z 576.75.

¹ H NMR (DMSO-d ₆ , δ, ppm, J / Hz): 0.46 (m, 1H, CH ₂ CH ₂ C H ₂ CH ₂ COOH), 0.75 (m, 1H, CH ₂ CH ₂ C H ₂ CH ₂ COOH), 1.23-1.33 (m, 8H, CH ₂ C H ₃ , CH ₂ C H ₂ CH ₂ CH ₂ COOH), 1.68 (s, 9H, C (3) H ₃ , C (3 ' , 3 ') H ₃ ), 1.89 (m, 2H, C H ₂ CH ₂ CH ₂ CH ₂ COOH), 2.16 (m, 1H, CH ₂ CH ₂ CH ₂ C H ₂ COOH), 2.4 (m, 1H, CH ₂ CH ₂ CH ₂ C H ₂ COOH), 4.13 (m, 4H, C H ₂ CH ₃ ), 6.26 (d, 1H, α'- CH , J = 13.5), 6.34 (d, 1H, α- CH , J = 13.5), 6.56 (t, 1H, γ- CH , J = 12.0), 7.2-7.8 (m, 7H, Ar H ), 8.35 (t, 2H, β, β'-CH, J = 13.0)

Example 30. Obtaining 3- (4-carboxybutyl) -3,3 ', 3'-trimethyl-5-sulfo-N, N’-diethyl-indodicarbocyanine dye (compound A5).

3- (4-carboxybutyl) -3,3 ', 3'-trimethyl-5-sulfo-N, N'-diethyl-indodicarbocyanine dye (compound A5) is obtained analogously to compound A2 with the following reagent ratios: (1 step) 3- (4-carboxybutyl) -2,3-dimethyl-5-sulfo-N-ethylindoleninium (compound 8) (71 mg, 0.2 mmol), malonic dialdehyde dianyl hydrochloride (compound 25) (52 mg, 0.2 mmol), acetic anhydride ( 700 μl), acetic acid (350 μl); (2 stage) 2,3,3-trimethyl-N-ethylindoleninium iodide (compound 23) (66 mg, 0.21 mmol), anhydrous potassium acetate (98 mg, 1 mmol), acetic anhydride (700 μl), acetic acid (350 μl). The reaction product is isolated analogously to compound A1. 20 mg (18%) of compound A5 are obtained. Mass spectrum (MALDI) (C ₃₃ H ₄₀ N ₂ O ₅ S): Found m / z 577.3, calculated m / z 576.75.

¹ H NMR (DMSO-d ₆ , δ, ppm, J / Hz): 0.47 (m, 1H, CH ₂ CH ₂ C H ₂ CH ₂ COOH), 0.77 (m, 1H, CH ₂ CH ₂ C H ₂ CH ₂ COOH), 1.24-1.36 (m, 8H, CH ₂ C H ₃ , CH ₂ C H ₂ CH ₂ CH ₂ COOH), 1.7 (s, 9H, C (3) H ₃ , C (3 ' , 3 ') H _3), 1.9 (m, 2H, C H ₂ CH ₂ CH ₂ CH ₂ COOH), 2.16 (m, 1H, CH ₂ CH ₂ CH ₂ C H ₂ COOH), 2.42 (m, 1H, CH ₂ CH ₂ CH ₂ C H ₂ COOH), 4.13 (m, 4H, C H ₂ CH _3), 6.27 (d, 1H, α'- CH, J = 13.5), 6.35 (d, 1H, α- CH , J = 13.5), 6.57 (t, 1H, γ- CH , J = 12.0), 7.21-7.75 (m, 7H, ArH), 8.34 (t, 2H, β, β'-C H , J = 13)

Example 31. Obtaining 5- (2-carboxyethyl) -3,3,3 ', 3'-tetramethyl-N- (4-sulfonate-butyl) -N’-ethylindodicarbocyanine dye (compound B1)

5- (2-Carboxyethyl) -3,3,3 ', 3'-tetramethyl-N- (4-sulfonateobutyl) -N'-ethylindodicarbocyanine dye (compound B1) is obtained analogously to compound A1 in the following reagent ratios: (1 stage) 2,3,3-trimethyl-N-ethylindoleninium iodide (compound 23) (54 mg, 0.2 mmol), malonic dialdehyde dianyl hydrochloride (compound 25) (59 mg, 0.23 mmol), acetic anhydride (1 ml), acetyl chloride (64 μl); (2 stage) 5- (2-carboxyethyl) -2,3,3-trimethyl-N- (4-sulfonate-butyl) indoleninium (compound 19) (81 mg, 0.22 mmol), diisopropylethylamine (200 μl, 2.1 mmol), acetic anhydride (2.8 ml). Obtain 57 mg (48%) of compound B1. Mass spectrum (MALDI) (C ₃₄ H ₄₂ N ₂ O ₅ S): found m / z 591.0, calculated m / z 590.77.

¹ H NMR (DMSO-d ₆ , δ, ppm, J / Hz): 1.26 (t, 3H, J 7.0, CH ₂ C H ₃ ), 1.63 (s, 12H, C (3.3) N ₃ , C (3 ', 3') H ₃ ), 1.73 (m, 4H, CH ₂ C H ₂ C H ₂ CH ₂ SO ₃ ), 2.48 (m, 2H, CH ₂ CH ₂ CH ₂ C H ₂ SO ₃ ), 2.58 (t, 2H, J 7.5, CH ₂ C H ₂ COOH, J = 7.5), 2.88 (t, 2H, C H ₂ CH ₂ COOH, J = 7.5), 4.10 (m, 4H, C H ₂ CH ₃ , C H ₂ CH ₂ CH ₂ CH ₂ SO ₃ ), 6.37 (d, 2H, α, α'-C H , J = 13.5), 6.56 (t, 1H, γ- CH , J = 12.0) , 7.19-7.63 (br.m., 7H, Ar H ), 8.31 (m, 2H, β, β'- СН )

Example 32. Obtaining 5- (2-carboxyethyl) -3,3,3 ', 3'-tetramethyl-N ’- (4-sulfonate-butyl) -N-ethylindodicarbocyanine dye (compound B2)

5- (2-Carboxyethyl) -3,3,3 ', 3'-tetramethyl-N' - (4-sulfonate-butyl) -N-ethylindodicarbocyanine dye (compound B2) is obtained analogously to compound A2 in the following reagent ratios: (1 stage) 2,3,3-trimethyl-N- (4-sulfonatobutyl) indoleninium (compound 22) (59 mg, 0.2 mmol), malonic dialdehyde dianyl hydrochloride (compound 25) (59 mg, 0.23 mmol), acetic anhydride (1.5 ml ), acetic acid (0.5 ml); (2 stage) 5- (2-carboxyethyl) -2,3,3-trimethyl-N-ethylindoleninium iodide (compound 18) (85 mg, 0.22 mmol), anhydrous potassium acetate (120 mg, 1.2 mmol), acetic anhydride ( 1.5 ml), acetic acid (0.5 ml). Obtain 59 mg (50%) of compound B2. Mass spectrum (MALDI) (C ₃₄ H ₄₂ N ₂ O ₅ S): found m / z 592.8, calculated m / z 590.77.

¹ H NMR (DMSO-d ₆ , δ, ppm, J / Hz): 1.28 (t, 3H, J 7.0, CH ₂ C H ₃ ), 1.67 (s, 12H, C (3.3) N ₃ , C (3 ', 3') H ₃ ), 1.74 (m, 4H, CH ₂ C H ₂ C H ₂ CH ₂ SO ₃ ), 2.48 (m, 2H, CH ₂ CH ₂ CH ₂ C H ₂ SO ₃ ), 2.58 (t, 2H, J 7.5, CH ₂ C H ₂ COOH, J = 7.5), 2.88 (t, 2H, C H ₂ CH ₂ COOH, J = 7.5), 4.13 (m, 4H, C H ₂ CH ₃ , C H ₂ CH ₂ CH ₂ CH ₂ SO ₃ ), 6.30 (d, 2H, α, α'-C H , J = 13.5), 6.56 (t, 1H, γ-C H , J = 12.0 ), 7.24-7.58 (m, 6H, Ar H ), 8.28 (m, 2H, β, β'-C H )

Example 33. Obtaining 5- (2-carboxyethyl) -3,3,3 ', 3'-tetramethyl-5'-sulfo-N, N'-diethylindodicarbocyanine dye (compound B3)

5- (2-Carboxyethyl) -3,3,3 ', 3'-tetramethyl-5'-sulfo-N, N'-diethylindodicarbocyanine dye (compound B3) is obtained analogously to compound A2 in the following ratio of reactants: (1 stage) , 3,3-trimethyl-5-sulfo-N-ethylindoleninium (compound 19) (53 mg, 0.2 mmol), malonic dialdehyde dianyl hydrochloride (compound 25) (59 mg, 0.23 mmol), acetic anhydride (1.5 ml), acetic acid (0.5 ml); (Stage 2) 5- (2-carboxyethyl) -2,3,3-trimethyl-N-ethylindoleninium iodide (compound 18) (85 mg, 0.22 mmol), anhydrous potassium acetate (120 mg, 1.2 mmol), acetic anhydride (1.5 ml), acetic acid (0.5 ml). 60 mg (53%) of compound B3 are obtained. Mass spectrum (MALDI) (C ₃₂ H ₃₈ N ₂ O ₅ S): found m / z 563.2, calculated m / z 562.72.

¹ H NMR (DMSO-d ₆ , δ, ppm, J / Hz): 1.25 (t, 6H, CH ₂ C H ₃ , J = 7.0), 1.69 (s, 12H, C (3.3) H ₃ , C (3 ', 3') H ₃ ), 2.32 (t, 2H, CH ₂ C H ₂ COOH, J = 7.5), 2.86 (t, 2H, C H ₂ CH ₂ COOH, J = 7.5) , 4.95 (m, 4H, C H ₂ CH ₃ ), 6.30 (d, 2H, α, α'- CH , J = 13.5), 6.58 (t, 1H, γ-C H , J = 12.0), 7.21- 7.63 (m, 6H, Ar H ), 8.31 (m, 2H, β, β'-C H )

Example 34. p-Nitrophenyl esters of dyes of General formulas (A) and (B).

A mixture of dye (0.044 mmol) and bis (p-nitrophenyl) carbonate (26 mg, 0.087 mmol) was dissolved in DMF (1 ml) and stirred for 2 h at room temperature. Then the reaction mass was diluted with water (10 ml) and the precipitate formed was removed by filtration. 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 35. Synthesis of fluorescently-labeled 5- (3-aminoallyl) -2′-deoxyuridine-5′-triphosphates.

To a solution of AAdUTP (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 A or B (2 μmol) in DMF (0.2 ml ) and the mixture was stirred for 12 hours at + 5 ° C. Conjugates of the general formula I were purified from excess dye A or 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 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 0.025 M to 0.3 M TEANS (pH 8.5 ) in 30% acetonitrile at a rate of 58 ml / h. The target substance was collected from the column in the concentration range of NH ₄ HCO ₃ from 0.2 to 0.3 M.

The fraction containing the modified nucleotide was collected, diluted to a volume of 50 ml with 0.1 M TEANS buffer solution (pH 8.5) and applied to a chromatography 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 eluted with a mixture of acetonitrile - water.

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

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

Example 36. Preparation of 5- [4-aza-5-oxo-9- (3,3 ', 3'-trimethyl-1- (4-sulfonate-butyl) -1'-ethylindo dicarbocyanin-3-yl) -non-1 en-1-yl)] - 2'-deoxyuridin-5'-triphosphate (compound TA1).

Obtain 0.56 μmol (35%) of compound TA1. Found: m / z 1111.6 [M] ⁺ . C ₄₇ H ₅₈ N ₅ O ₁₈ P ₃ S ^4- . Calculated: M = 1105.97.

¹ H NMR (D ₂ O, δ, ppm, J / Hz): 0.47, 0.76 (2 m, 2H, dye C (8) H ₂ ), 1.22 (t, 3H, CH ₂ C H ₃ , J = 7.0), 1.31 (m, 2H, dye C (9) H ₂ ), 1.64 (s, dye 9H, C (3) H ₃ , C (3 ', 3') H ₃ ), 1.77 (m, 4H, CH ₂ C H ₂ C H ₂ CH ₂ SO ₃ ), 1.92 (m, 2H, dye C (7) H ₂ ), 2.21 (m, 6H, C (2 ') H ₂ , dye C (6) H ₂ , CH ₂ CH ₂ CH ₂ C H ₂ SO ₃ ), 3.80 (m, 2H, dye C (3) H ₂ ), 4.08 (br.m, 8H, C (3 ') H , C (4 ') H , C (5') H ₂ , C H ₂ CH ₃ , C H ₂ CH ₂ CH ₂ CH ₂ SO ₃ ), 6.19 (m, 3H, dye α, α'-C H , C (1 ' ) H ), 6.34 (m, 2H, dye C (1) H , C (2) H ), 6.41 (m, 1H, dye γ-C H ), 7.15, 7.21, 7.38, 7.61, 7.71 (5 m. 9H, C (6) H , Ar H ), 7.95 (m, 2H, dye β, β'-C H )

NMR ³¹ P (D ₂ O, δ, ppm): -21.16 (t, ^β P), -11.06 (d, ^α P), -7.98 (d, ^γ P)

Example 37. Preparation of 5- [4-aza-5-oxo-9- (3,3 ', 3'-trimethyl-1,1'-di- (4-sulfonate-butyl)) indodicarbocyanin-3-yl) nonon- 1-en-1-yl)] - 2'-deoxyuridin-5'-triphosphate (compound TA2).

Obtain 0.72 μmol (46%) of compound TA2. Found: m / z 1219.3 [M] ⁺ . C ₄₉ H ₆₁ N ₅ O ₂₁ P ₃ S ₂ ^5- . Calculated: M = 1213.08.

¹ H NMR (D ₂ O, δ, ppm, J / Hz): 0.45, 0.77 (2 m, 2H, dye C (8) H ₂ ), 1.35 (m, 2H, dye C (9) N ₂ ), 1-62 (s, dye 9Н, С (3) Н ₃ , С (3 ', 3') Н ₃ ), 1.75 (m, 4Н, CH ₂ C H ₂ C H ₂ CH ₂ SO ₃ ) , 1.9 (m, 2H, dye C (7) H ₂ ), 2.25 (m, 6H, C (2 ') H ₂ , dye C (6) H ₂ , CH ₂ CH ₂ CH ₂ C H ₂ SO ₃ ) , 3.85 (m, 2H, dye C (3) H ₂ ), 4.12 (br.m, 8H, C (3 ') H , C (4') H , C (5 ') H ₂ , C H ₂ CH ₂ CH ₂ CH ₂ SO ₃ ), 6.15 (m, 3H, dye α, α'-С Н , С (1 ') Н ), 6.31 (m, 2Н, dye С (1) Н , С (2) Н ), 6.39 (m, 1H, dye γ-С Н ), 7.11, 7.25, 7.41, 7.59, 7.64 (5 m, 9Н, С (6) Н , Ar H ), 8.08 (m, 2Н, dye β, β'-C H )

NMR ³¹ P (D ₂ O, δ, ppm): -22.86 (t, ^β P), -11.94 (d, ^α P), -7.05 (d, ^γ P)

Example 38. Obtaining 5- [4-aza-5-oxo-9- (3,3 ', 3'-trimethyl-1' - (4-sulfonate-butyl) -1-ethylindodicarbocyanin-3-yl) -non-1- en-1-yl)] - 2'-deoxyuridin-5'-triphosphate (compound TA3).

Obtain 0.62 μmol (39%) of compound TA3. Found: m / z 1111.2 [M] ⁺ . C ₄₇ H ₅₈ N ₅ O ₁₈ P ₃ S ^4- . Calculated: M = 1105.97.

¹ H NMR (D ₂ O, δ, ppm, J / Hz): 0.45, 0.78 (2 m, 2H, dye C (8) H ₂ ), 1.21 (t, 3H, CH ₂ C H ₃ , J = 7.0), 1.34 (m, 2H, dye C (9) H ₂ ), 1.62 (s, dye 9H, C (3) H ₃ , C (3 ', 3') H ₃ ), 1.72 (m, 4H, CH ₂ C H ₂ C H ₂ CH ₂ SO ₃ ), 1.91 (m, 2H, dye C (7) H ₂ ), 2.24 (m, 6H, C (2 ') H ₂ , dye C (6) H ₂ , CH ₂ CH ₂ CH ₂ C H ₂ SO ₃ ), 3.81 (m, 2H, dye C (3) H ₂ ), 4.12 (br.m, 8H, C (3 ') H , C (4' ) Н , С (5 ') Н ₂ , С Н ₂ СН ₃ , C H ₂ CH ₂ CH ₂ CH ₂ SO ₃ ), 6.12 (m, 3H, dye α, α'-С Н , С (1') H ), 6.32 (m, 2H, dye C (1) H , C (2) H ), 6.45 (m, 1H, dye γ-C H ), 7.12, 7.25, 7.38, 7.65, 7.75 (5 m, 9H, C (6) H , Ar H ), 7.99 (m, 2H, dye β, β'-C H )

NMR ³¹ P (D ₂ O, δ, ppm): -21.56 (t, ^β P), -11.26 (d, ^α P), -7.13 (d, ^γ P)

Example 39. Obtaining 5- [4-aza-5-oxo-9- (3,3 ', 3'-trimethyl-5'-sulfo-1,1'-diethyl-indodicarbocyanin-3-yl) -non-1 en-1-yl)] - 2'-deoxyuridin-5'-triphosphate (compound TA4).

Obtain 0.78 μmol (49%) of compound TA4. Found: m / z 1083.1 [M] ⁺ . C ₄₅ H ₅₄ N ₅ O ₁₈ P ₃ S ^4- . Calculated: M = 1077.92.

¹ H NMR (D ₂ O, δ, ppm, J / Hz): 0.43, 0.72 (2 m, 2H, dye C (8) H ₂ ), 1-28 (m, 6H, CH ₂ C N ₃ ), 1.35 (m, 2H, dye C (9) H ₂ ), 1.58 (s, dye 9H, C (3) H ₃ , C (3 ', 3') H ₃ ), 1.88 (m, 2H, dye C (7) H ₂ ), 2.24 (m, 4H, C (2 ') H ₂ , dye C (6) H ₂ ), 3.85 (m, 2H, dye C (3) H ₂ ), 4.13 (ear m, 8Н, С (3 ') Н , С (4') Н , С (5 ') Н ₂ , С Н ₂ СН ₃ ), 6.21 (m, 3H, dye α, α'-С Н , С (1 ') H ), 6.31 (m, 2H, dye C (1) H , C (2) H ), 6.45 (m, 1H, dye γ-C H ), 7.14, 7.19, 7.31, 7.58, 7.68 ( 5 m, 8Н, С (6) Н , Ar H ), 7.98 (m, 2Н, dye β, β'-С Н )

NMR ³¹ P (D ₂ O, δ, ppm): -22.35 (t, ^β P), -12.64 (d, ^α P), -8.68 (d, ^γ P)

Example 40. Preparation of 5- [4-aza-5-oxo-9- (3,3 ', 3'-trimethyl-1,1'-diethyl-5-sulfo-indodicarbocyanin-3-yl) non-1- en-1-yl)] - 2'-deoxyuridin-5'-triphosphate (compound TA5).

Obtain 0.67 μmol (42%) of compound TA5. Found: m / z 1083.4 [M] ⁺ . C ₄₅ H ₅₄ N ₅ O ₁₈ P ₃ S ^4- . Calculated: M = 1077.92.

¹ H NMR (D ₂ O, δ, ppm, J / Hz): 0.44, 0.71 (2 m, 2H, dye C (8) H ₂ ), 1.25 (m, 6H, C H ₂ CH ₃ ) , 1.37 (m, 2H, dye C (9) H ₂ ), 1.59 (s, dye 9H, C (3) H ₃ , C (3 ', 3') H ₃ ), 1.89 (m, 2H, dye C (7) H ₂ ), 2.26 (m, 4H, C (2 ') H ₂ , dye C (6) H ₂ ), 3.83 (m, 2H, dye C (3) H ₂ ), 4.08 (br.m , 8H, С (3 ') Н , С (4') Н , С (5 ') Н ₂ , С Н ₂ СН ₃ ), 6.23 (m, 3H, dye α, α'-С Н , С (1 ') H ), 6.33 (m, 2H, dye C (1) H , C (2) H ), 6.43 (m, 1H, dye γ-CH), 7.15, 7.17, 7.3, 7.56, 7.66 (5 m. , 8Н, С (6) Н , Ar H ), 7.99 (m, 2Н, dye β, β'-С Н )

NMR ³¹ P (D ₂ O, δ, ppm): -22.15 (t, ^β P), -12.54 (d, ^α P), -8.24 (d, ^γ P)

Example 41. Preparation of 5- [4-aza-5-oxo-7- (1- (4-sulfonate-butyl) -3,3,3 ', 3'-tetramethyl-1'-ethylindodicarbocyanine-5-yl) -hept- 1-en-1-yl)] - 2'-deoxyuridin-5'-triphosphate (compound TB1).

Obtain 1 μmol (63%) of compound TB1. Found: m / z 1092.4 [M] ⁺ . C ₄₆ H ₅₆ N ₅ O ₁₈ P ₃ S ₂ ^4- . Calculated: M = 1091.95.

¹ H NMR (D ₂ O, δ, ppm, J / Hz): 1.24 (t, 3H, CH ₂ C H ₃ , J = 7.0), 1.59 (s, dye 12H, C (3.3) H ₃ , C (3 ', 3') H ₃ ), 1.73 (m, 4H, CH ₂ C H ₂ CH ₂ CH ₂ SO ₃ ), 2.21 (m, 4H, C (2 ') H ₂ , CH ₂ CH ₂ CH ₂ C H ₂ SO ₃ ), 2.52 (t, 2H, dye C (6) H ₂ , J = 7.5), 2.91 (t, 2H, dye C (7) H ₂ , J = 7.5), 3.82 (m, 2H, dye C (3) H ₂ ), 4.08 (br.m, 8H, C (3 ') H , C (4') H , C (5 ') H ₂ , C H ₂ CH ₃ , C H ₂ CH ₂ CH ₂ CH ₂ SO ₃ ), 6.21 (m, 3H, dye α, α'-С Н , С (1 ') H ), 6.29 (m, 2Н, dye С (1) Н , С (2) H ), 6.42 (m, 1H, dye γ-C H ), 7.25, 7.32, 7.39, 7.62, 7.71 (5 m, 8H, C (6) H , Ar H ), 8.07 (m, 2H dye β, β'-C H )

NMR ³¹ P (D ₂ O, δ, ppm): -21.45 (t, ^β P), -11.03 (d, ^α P), -7.97 (d, ^γ P)

Example 42. Preparation of 5- [4-aza-5-oxo-7- (1 '- (4-sulfonate-butyl) -3,3,3', 3'-tetramethyl-1-ethyline to dicarbocyanin-5-yl) - hept-1-en-1-yl)] - 2'-deoxyuridin-5'-triphosphate (compound TB2).

Obtain 0.93 μmol (58%) of compound TB2. Found: m / z 1093.6 [M] ⁺ . C ₄₆ H ₅₆ N ₅ O ₁₈ P ₃ S ₂ ^4- . Calculated: M = 1091.95.

¹ H NMR (D ₂ O, δ, ppm, J / Hz): 1.22 (t, 3H, CH ₂ C H ₃ , J = 7.0), 1.62 (s, dye 12H, C (3.3) H ₃ , C (3 ', 3') H ₃ ), 1.76 (m, 4H, CH ₂ C H ₂ C H ₂ CH ₂ SO ₃ ), 2.25 (m, 4H, C (2 ') H ₂ , CH ₂ CH ₂ CH ₂ C H ₂ SO ₃ ), 2.56 (t, 2H, dye C (6) H ₂ , J = 7.5), 2.92 (t, 2H, dye C (7) H ₂ , J = 7.5), 3.80 (m, 2H, dye C (3) H ₂ ), 4.12 (br.m, 8H, C (3 ') H , C (4') H , C (5 ') H ₂ , C H ₂ CH ₃ , C H ₂ CH ₂ CH ₂ CH ₂ SO ₃ ), 6.17 (m, 3H, dye α, α'-С Н , С (1 ') Н ), 6.32 (m, 2Н, dye С (1) Н , C (2) H ), 6.42 (m, 1H, dye γ-C H ), 7.23, 7.29, 7.41, 7.69, 7.74 (5 m, 8H, C (6) H , Ar H ), 7.98 (m, 2H, dye β, β'-C H )

NMR ³¹ P (D ₂ O, δ, ppm): -22.84 (t, ^β P), -11.47 (d, ^α P), -7.13 (d, ^γ P)

Example 43. Obtaining 5- [4-aza-5-oxo-7- (3,3,3 ', 3'-tetramethyl-5'-sulfo-1,1'-diethylindodicarbocyanin-5-yl) -hept-1 en-1-yl] -2'-deoxyuridin-5'-triphosphate (compound TB3).

Obtain 1.2 μmol (77%) of compound TB3. Found: m / z 1064.58 [M] ⁺ . C ₄₄ H ₅₂ N ₅ O ₁₈ P ₃ S ^4- . Calculated: M = 1063.89.

¹ H NMR (D ₂ O, δ, ppm, J / Hz): 1.25 (t, 3H, CH ₂ C H ₃ , J = 7.0), 1.68 (s, dye 12H, C (3.3) H ₃ , C (3 ', 3') H ₃ ), 2.21 (m, 2H, C (2 ') H ₂ ), 2.53 (t, 2H, dye C (6) H ₂ , J = 7.5), 2.78 (t, 2H, dye C (7) H ₂ , J = 7.5), 3.82 (m, 2H, dye C (3) H ₂ ), 4.08 (br.m, 8H, C (3 ') H , C ( 4 ') H , C (5') H ₂ , C H ₂ CH ₃ ), 6.19 (m, 3H, dye α, α'-C H , C (1 ') H ), 6.35 (m, 2H, dye C (1) H , C (2) H ), 6.45 (m, 1H, dye γ-C H ), 7.15, 7.21, 7.36, 7.61, 7.72 (5 m, 7H, C (6) H , Ar H ), 7.95 (m, 2H, dye β, β'-С Н )

NMR ³¹ P (D ₂ O, δ, ppm): -23.15 (t, ^β P), -10.85 (d, ^α P), -7.89 (d, ^γ P)

Example 44. The use of fluorescently-labeled nucleoside triphosphates of the general formula (I) using the TB-biochip test system as an example.

DNA samples were analyzed in accordance with the manufacturer's recommendations (Biochip-IMB, Moscow, Russia). Amplification of the DNA sample was carried out by the method of multiplex two-round PCR. The detection of PCR products after each step was confirmed by agarose gel electrophoresis. At the first stage of multiplex PCR, genomic DNA (3 μl) was amplified in a final volume of 30 μl. The reaction mixture (final volume 30 μl) contained 3 μl Taq buffer, 2.5 units. Taq polymerase, 0.2 μM of each deoxynucleoside triphosphate, primers, and 3 μl of DNA sample. The temperature regime was as follows: denaturation 95 ° C - 4 min, then 36 cycles of amplification (95 ° C - 30 sec, 67 ° C - 30 sec, 72 ° C - 30 sec), the final stage of incubation 72 ° C - 5 min. As a matrix for conducting 2 stages used 1 μl of the reaction mixture obtained in stage 1. 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 8 μmol of fluorescently labeled nucleotide and an excess of one primer. The temperature regime was as follows: denaturation 95 ° C for 5 min, then 37 cycles of amplification (95 ° C for 20 sec, 65 ° C for 30 sec, 72 ° C for 30 sec), the final incubation stage 72 ° C for 5 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 h. 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 bio-analyzer (Biochip-IMB, Russia) using specialized Imageware software.

Example 45. The use of fluorescently labeled nucleoside triphosphates of the general formula (I) is exemplified by the PF Biochip Trombo test system.

DNA samples were analyzed in accordance with the manufacturer's recommendations (Biochip-IMB, Moscow, Russia). Amplification of the DNA sample was carried out by the method of multiplex two-round PCR. The detection of PCR products after each step was confirmed by agarose gel electrophoresis. The reaction mixture (final volume 25 μl) contained 10x PCR buffer, 2.5 units. Taq polymerase, 0.2 μM of each deoxynucleoside triphosphate, 0.4 picamole primers, and 10 ng of DNA sample. The temperature regime was as follows: denaturation 94 ° C - 4 min, then 35 amplification cycles (94 ° C - 30 sec, 62 ° C - 30 sec, 72 ° C - 30 sec), the final stage of incubation 72 ° C - 5 min. As a matrix for conducting 2 stages used 2 μl of the reaction mixture obtained in stage 1. 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 8 μmol of fluorescently labeled nucleotide and an excess of one primer. The temperature regime was as follows: denaturation 94 ° C - 4 min, then 35 amplification cycles (94 ° C - 30 sec, 62 ° C - 30 sec, 72 ° C - 30 sec), the final stage of incubation 72 ° C - 5 min. Hybridization of the fluorescently-labeled product was carried out in accordance with the manufacturer's recommendations (Biochip-IMB, Moscow, Russia).

Fluorescently-labeled nucleoside triphosphates of the general formula (I) can be used in all diagnostic test systems based on PCR followed by hybridization analysis on oligonucleotide biochips and fluorescence recording of analysis results.

Claims (11)

1. Compounds of 5- [4-aza-5-oxo-9- (3,3 ', 3'-trimethyl-1- (4-sulfonate-butyl) -1'-ethylindodicarbocyanin-3-yl) non-1-en-1- sludge]] - 2'-deoxyuridine-5'-triphosphate, 5- [4-aza-5-oxo-9- (3,3 ', 3'-trimethyl-1,1'-di- (4-sulfonate-butyl)) indodicarbocyanin-3-yl) non-1-en- 1-yl)] - 2'-deoxyuridin-5'-triphosphate, 5- [4-aza-5-oxo-9- (3,3 ', 3'-trimethyl-1' - (4-sulfonate-butyl) -1-ethylindodicarbocyanin-3-yl) non-1-en-1- sludge]] - 2'-deoxyuridine-5'-triphosphate, 5- [4-aza-5-oxo-9- (3,3 ', 3'-trimethyl-5'-sulfo-1,1'-diethyl-indodicarbocyanin-3-yl) non-1-en-1 -yl)] - 2'-deoxyuridine-5'-triphosphate, 5- [4-aza-5-oxo-9- (3,3 ', 3'-trimethyl-1,1'-diethyl-5-sulfo-indodicarbocyanin-3-yl) non-1-en-1- sludge]] - 2'-deoxyuridine-5'-triphosphate or their salts. 2. Compounds of 5- [4-aza-5-oxo-7- (1- (4-sulfonatobutyl) -3,3,3 ', 3'-tetramethyl-1'-ethylindodicarbocyanin-5-yl) -hept-1-en- 1-yl)] - 2'-deoxyuridin-5'-triphosphate, 5- [4-aza-5-oxo-7- (1 '- (4-sulfonate-butyl) -3,3,3', 3'-tetramethyl-1-ethylindodicarbocyanin-5-yl) -hept-1-en- 1-yl)] - 2'-deoxyuridin-5'-triphosphate, 5- [4-aza-5-oxo-7- (3,3,3 ', 3'-tetramethyl-5'-sulfo-1,1'-diethylindodicarbocyanin-5-yl) -hept-1-en-1 -yl] -2 '-deoxyuridine-5'-triphosphate or their salts. 3. The use of compounds according to claims 1 and 2 as fluorescent labels for DNA labeling during PCR.

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