RU2278869C2 - New substrates for terminal deoxynucleotidyl transferase - Google Patents

Abstract

FIELD: molecular biology, biotechnology, biochemistry, pharmaceutical chemistry.

SUBSTANCE: invention relates to new unnatural substrates for enzyme terminal deoxynucleotidyl transferase that can be used as a tool for insertion of label by 3'-hydroxyl group of oligodeoxynucleotides and single-stranded DNA. Invention describes new substrates for terminal deoxynucleotidyl transferase of the general formula (I):

wherein R represents a marker group chosen from dansyl, dinitrophenyl, fluorescein, fluorenylmethyloxycarbonyl, rhodamine B, tetramethylrhodamine, eosin, erythrosine, trioxalene, BODIPY, biotin, auramine, psoralene, pyrene, Texas red dye, actinomycin D, 7-methoxycoumarin and ellipticin; X is a linker representing a linear carbon chain that comprises heteroatoms, such as nitrogen and oxygen atoms, and this chain can comprise carbonyl and thiocarbonyl as functional groups. Invention provides preparing new effective substrate for enzyme.

EFFECT: valuable biochemical properties of substrates.

1 tbl, 2 dwg, 7 ex

Description

The present invention relates to the field of molecular biology, biotechnology and pharmaceutical chemistry. More specifically, the invention relates to new non-natural substrates of the terminal deoxynucleotidyl transferase (TDT) enzyme, which can be used as a 3'-hydroxyl labeling agent for oligodeoxynucleotides and single-stranded DNAs.

New substrates are analogues of nucleoside triphosphates containing marker groups instead of the nucleoside component.

Terminal deoxynucleotidyl transferase (TDT), a matrix-independent DNA polymerase, plays an important role in the formation of the human and animal immune systems. TDT is currently widely used in molecular biology. Natural substrates of this enzyme are deoxynucleoside triphosphates. However, it was previously shown [Arzumanov A.A., Victorova L.S., Jasko M.V. Synthesis of non-nucleoside triphosphate analog, a new type of substrates for terminal deoxynucleotidyl transferase, Nucleosides, Nucleotides & Nucleic Acids, 2000, 19, No. 10-12, 1787-1793], that the presence of a nucleoside fragment is not necessary for the manifestation of substrate properties in relation to TDT. A number of triphosphates were synthesized, containing instead of the nucleoside component various bulky substituents [Arzumanov A.A., Victorova L.S., Jasko M.V., Yesipov D.S., Krayevsky A.A. Terminal Deoxynucleotidyl Transferase Catalyzes the Reaction of DNA Phosphorylation., Nucl. Acids Res., 2000, 28, No. 5, 1276-1281]. It has been shown that compounds of this type are TDT substrates. The effectiveness of compounds such as TDT substrates depends on the structure of the bulky substituent introduced instead of the nucleoside fragment and on the length and structure of the linker [A.L. Khandazhinskaya, M.V. Jasko, E.A. Shirokova, M.K. Kukhanova "New substrates of terminal deoxynucleotidyl transferase: synthesis and biological evaluation" Collection Symposium Series. 2002, v 5, p. 344-347].

The introduction of marker groups into oligonucleotides is currently widely used in molecular biology and is carried out by chemical labeling or enzymatic addition of modified nucleotides. Despite this, the development of new methods for introducing marker groups into oligonucleotides remains a very urgent task. The approach developed in our laboratory allows the use of nucleotide-containing TDT substrates for introducing marker groups into oligonucleotides and single-stranded DNA, which is a fundamentally new way of introducing labels.

The authors of the present invention had the task of creating new effective TDT substrates and expanding the arsenal of such substrates, which is necessary to solve the problems of molecular biology, biotechnology and medicine.

The present invention allows the preparation of effective TDT substrates, which are triphosphates of the general formula I:

where R is a marker group;

X is a linker, which is a linear hydrocarbon chain, which may contain double and / or triple bonds, aromatic cycles; heteroatoms such as oxygen, sulfur, nitrogen; however, as functional groups, the chain may contain hydroxy-, amino-, amido-, carbonyl-, thiocarbonyl-, carboxy- and thiocarboxy- and aromatic groups.

The following can be used as marker groups: dansil, dinitrophenyl, fluorenylmethyloxycarbonyl, biotin, auramine, psoralen, pyrene, fluorescein, Texas red, actinomycin D, BODIPY, 7-methoxycoumarin, eosin, erythrosine, trioxalene, rhodamine B, any tetramethylrodin and tetramethylrod another known as a marker group.

Compounds of formula I are prepared according to one of two schemes. One of them consists in the initial preparation of triphosphate of the linker part of the molecule, followed by the introduction of a marker group. Another approach involves the attachment of a marker group to the linker and subsequent phosphorylation.

We have found that type I compounds bearing linkers with marker (reporter or ligand) groups instead of a nucleoside fragment are effective TDT substrates.

The mechanism of the enzymatic reaction involving compounds of type I is described by the following equation:

where (pdN) _n pdN is an oligonucleotide or single-stranded DNA

Figure 1 illustrates the substrate properties of compounds Ia and IB. Figure 1 shows the separation of the reaction products in a denaturing 15% polyacrylamide gel after incorporation of compounds Ia and IB at the 3'-end of the oligonucleotide upon catalysis by terminal deoxynucleotidyl transferase.

Figure 2 illustrates the substrate properties of compound IB. Figure 2 shows the separation of the reaction products in a denaturing 15% polyacrylamide gel after incorporation of compound IB into the 3'-end of the oligonucleotide by terminal deoxynucleotidyl transferase.

The invention is illustrated by the following examples.

Example 1. Obtaining N- (5-N, N-dimethylaminonaphthalene-1-sulfonyl) -aminobutyl triphosphate (Ia).

The synthesis of compound Ia was carried out according to the following scheme:

where the DNS-danced radical of the formula:

1.1. N- (5-N, N-dimethylaminonaphthalene-1-sulfonyl) aminobutanol

A mixture of aminobutanol (178 mg, 1 mmol) and 5-N, N-dimethylaminonaphthalene-1-sulfonyl chloride (135 mg, 0.5 mmol) in 3 ml of dioxane was stirred for 20 hours at 20 ° C, then evaporated and the product was isolated on a column ( 2 × 17 cm) with silica gel (Merck). Elution was carried out in portions of 100 ml of chloroform, then with 1, 2 and 3% MeOH in chloroform. The product was eluted with 4% MeOH in chloroform and evaporated. Yield 67 mg (41%).

UV (MeOH) λmax 337 (ε 2500), λmax 250 (ε 8670), λmax 219 (ε 21700).

¹ H NMR (CDCl ₃ ): 1.40 m (4H, (CH ₂ ) ₂ center.), 2.91 m (8H, 2CH ₃ + CH ₂ N), 3.50 t (2H, J 5.9 , CH ₂ O), 5.21 t (1H, J 5.9, NH), 7.17 d (1H, J 7.48, H-6), 7.53 m (2H, H-3 + H -7), 8.21 d (1H, J 7.16, H-8), 8.23 d (2H, J 8.41, H-4), 8.29 d (1H, J 8.4, H-2).

1.2. N- (5-N, N-dimethylaminonaphthalene-1-sulfonyl) aminobutyl ester of phosphoric acid

To a solution of 1,2,4-triazole (35 mg, 500 μmol) in 1 ml of abs. CH ₃ CN and triethylamine (80 μl, 500 μmol) were added at -10 ° C POCl ₃ (15.8 μl, 170 μmol), the mixture was kept for 40 min at -10 ° C and centrifuged. The supernatant was added to N- (5-N, N-dimethylaminonaphthalen-1-sulfonyl) aminobutanol (18 mg, 56.7 μmol), pre-dried by 3-fold evaporation with abs. pyridine. After 2 hours at 20 ° C, the mixture was diluted with water (50 ml), kept for 1 hour and applied to a column (2 × 9 cm) with a DEAE-Toyopearl 650 (Toyo Soda, Japan). Eluted in a linear gradient of NH ₄ HCO ₃ concentrations (0 → 0.4 M) in 10% MeOH (eluent volume 0.6 L). The fractions containing the product were evaporated, re-evaporated with water (10 × 10 ml), the residue was lyophilized. Yield 11.8 mg (27 μmol, 47%).

UV (water) λmax 333 (ε 2500), λmax 245 (ε 8670), λmax 216 (ε 21700).

¹ H NMR (D ₂ O): 1.14 m (4H, (CH ₂ ) ₂ center.), 2.87 m (2H CH ₂ N), 3.03 m (6H, (CH ₃ ) ₂ )) 3.50 m (2H, CH ₂ O), 7.43 d (1H, J 7.78, H-6), 7.58 m (2H, H-3 + H-7), 8.14 d (1H, J 7.16, H-8), 8.29 d (2H, J 8.41, H-4), 8.34 d (1H, J 8.4, H-2). ³¹ P NMR (D ₂ O) 2.1 s.

1.3. N- (5-N, N-dimethylaminonaphthalene-1-sulfonyl) -4-aminobutyl ester of triphosphoric acid

To a solution of the triethylammonium salt of N- (5-N, N-dimethylaminonaphthalene-1-sulfonyl) aminobutyl phosphate (15.1 mg, 25 μmol) in abs. DMF (3 ml) was added N, N'-carbonyldiimidazole (CDI) (32 mg, 0.2 mmol). The reaction mixture was stirred for 18 hours at 4 ° C under argon, then MeOH (0.33 ml, 8 mmol) was added and left for 40 min at 20 ° C. The reaction mixture was concentrated to 2 ml and 0.3 M (Bu ₃ NH) ₂ H ₂ P ₂ O ₇ in DMF (0.5 ml) was added. The reaction mixture was stirred for 18 h at 20 ° C, then diluted with water to 10 ml and applied onto a Dowex 50 column (2 × 5 cm) in NH ₄ ⁺ form, eluting with water. The UV absorbing fractions were evaporated, the residue was dissolved in 0.3 ml of water and applied onto a LiChroprep RP 8 column (1 × 15 cm) in 5 mM NH ₄ HCO ₃ . Elution was carried out in a linear gradient of MeOH concentrations (0 → 20%) in 5 mM NH ₄ HCO ₃ . Fractions containing the desired product were evaporated, re-evaporated with water (2 × 5 ml) and lyophilized. Yield 5.9 mg (9.3 μmol, 37%).

UV (water) λmax 333 (ε 2500), λmax 245 (ε 8670), λmax 216 (ε 21700).

¹ H NMR (D ₂ O): 1.27 m (4H, (CH ₂ ) ₂ center.), 2.74 s (6H, (CH ₃ ) ₂ ), 2.81 m (2H CH ₂ N), 3.59 m (2H, CH ₂ O), 7.29 d (1H, J 7.78, H-6), 7.56 m (2H, H-3 + H-7), 8.12 d ( 1H, J 7.16, H-8), 8.33 d (1H, J 8.41, H-4), 8.37 d (1H, J 8.4, H-2)

³¹ P NMR (D ₂ O) -7.4 d (J _Pα , _Pβ 19.5 P _α ), -10.6 d (J _{Pβ, Pγ} 20.3 P _γ ), -22.2 t (P _β )

Example 2. Obtaining N - [(5-N, N-dimethylaminonaphthalene-1-sulfonyl) aminohexanoyl] -2-aminoethyl ester of triphosphoric acid (IB).

The synthesis of compound IB was carried out according to the following scheme:

2.1. N - [(5-N, N-dimethylaminonaphthalene-1-sulfonyl) aminohexanoyl] -2-aminoethyl ester of phosphoric acid

To a solution of N - [(5-N, N-dimethylaminonaphthalene-1-sulfonyl) aminohexanoic acid (54 mg, 0.15 mmol) in 3 ml of abs. DMF was added CDI (48 mg, 0.3 mmol). The reaction mixture was stirred for 1 h at 20 ° C, then added to a solution of aminoethyl phosphate (28 mg, 0.2 mmol) in 3 ml of water and 20 μl of triethylamine. After 18 hours at 20 ° C, the mixture was diluted with water (50 ml) and applied to a DEAE-Toyopearl 650 column (2 × 9 cm). Eluted in a linear gradient of concentrations of NH ₄ HCO ₃ (0 → 0.4 M, 0.6 L) in 10% MeOH. The fractions containing the product were evaporated, re-evaporated with water (10 × 10 ml), the residue was lyophilized. Yield 49.5 mg (95 μmol, 63%).

UV (water) λmax 333 (ε 2500), λmax 245 (ε 8670), λmax 216 (ε 21700).

¹ H NMR (D ₂ O): 0.92 m (2H, -CH ₂ center), 1.08 m (4H, 2 (CH ₂ )), 1.80 t (2H, 7.5 Hz, CH _z N), 2,84 s (6H, N (CH _{3) 2),} 2.89 m (2H, 6.53 Hz, CH ₂ O), 3,20 m (2H, 7.17 Hz, CH ₂ NH), 3.26 t (2H, 4.78 Hz, CH ₂ O), 7.39 d (1H, J 7.79, H-6), 7.66 m (2H, H-3 + H -7), 8.22 d (1H, J 6.54, H-8), 8.24 d (2H, J 8.72, H-4), 8.47 d (1H, J 8.72, H-2). ³¹ P NMR (D ₂ O) 4.5 s.

2.2. N - [(5-N, N-dimethylaminonaphthalene-1-sulfonyl) aminohexanoyl] -2-aminoethyl ester of triphosphoric acid

To a solution of the triethylammonium salt of 2-N - [(5-N, N-dimethylaminonaphthalen-1-sulfonyl) aminohexanoyl] aminoethyl phosphate (17.2 mg, 25 μmol) in 3 ml of abs. DMF was added carbonyldiimidazole (32 mg, 0.2 mmol). The reaction mixture was stirred for 18 hours at 4 ° C under argon, then MeOH (0.33 ml, 8 mmol) was added and left for 40 min at 20 ° C. The reaction mixture was concentrated to 2 ml and a 0.3 M solution of (Bu ₃ NH) ₂ H ₂ P ₂ O ₇ (0.5 ml, 150 μmol) was added. The reaction mixture was stirred for 18 hours at 20 ° C, then diluted with water to 10 ml and applied to a Dowex 50 column (2 × 5 cm) in NH ₄ ⁺ form. The substance was eluted with water. The UV absorbing fractions were evaporated, the residue was dissolved in 0.3 ml of water and applied to a LiChroprep RP-8 column (1 × 15 cm) in 5 mM NH ₄ HCO ₃ . Elution was carried out in a linear gradient of MeOH concentrations (0 → 20%) in 5 mM NH ₄ HCO ₃ . Fractions containing the desired product were evaporated, evaporated with water (2 × 5 ml) and lyophilized. Yield 6.5 mg (9.3 μmol, 37%).

UV (water) λmax 333 (ε 2500), λmax 245 (ε 8670), λmax 216 (ε 21700).

¹ H NMR (D ₂ O): 0.92 m (2H, —CH ₂ center), 1.08 m (4H, 2 (CH ₂ )), 1.80 t (2H, 7.47 Hz, CH ₂ NH), 2.84 s (6H, N (CH ₃ ) ₂ ), 2.89 t (2H, 6.53 Hz, CH ₂ O), 3.20 t (2H, 7.17 Hz, CH ₂ NH), 3.26 t (2H, 4.78 Hz, CH ₂ O), 7.39 d (1H, J 7.79, H-6), 7.66 m (2H, H-3 + H-7 ), 8.22 d (1H, J 6.54, H-8), 8.24 d (2H, J 8.72, H-4), 8.47 d (1H, J 8.72, H- 2). ³¹ P NMR (D ₂ O) -6.4 d (J _{Pα, Pβ} 21.3 P _α ), -10.3 d (J _{Pβ, Pγ} 19.3 P _γ ), -21.8 t (P _β )

Example 3. Obtaining N- [6-N- (2,4-dinitrophenyl) aminohexanoyl] -2-aminoethyl ester of triphosphoric acid (Ic)

The synthesis of compound IB was carried out according to the following scheme:

3.1. N- [6-N- (2,4-Dinitrophenyl) aminohexanoyl] -2-aminoethyl ester of phosphoric acid

To a solution of 6-N- (2,4-dinitrophenyl) aminohexanoic acid (44 mg, 0.15 mmol) in 3 ml abs. DMF was added CDI (48 mg, 0.3 mmol). The reaction mixture was stirred for 1 h at 20 ° C, then added to a solution of aminoethyl phosphate (28 mg, 0.2 mmol) in 3 ml of water and 20 μl of triethylamine. After 18 h at 20 ° С, the mixture was diluted with water (50 ml) and applied onto a DEAE-Toyopearl 650 column (2 × 9 cm), eluting with a linear concentration gradient of NH ₄ НСО ₃ (0 → 0.4 М, 0.6 L) ) in 10% MeOH. The fractions containing the product were evaporated, re-evaporated with water (10 × 10 ml), the residue was lyophilized. Yield 44.8 mg (0.1 mmol, 67%).

UV (water) λmax 363 (ε 17500), λmax 265 (ε 8340).

¹ H NMR (D ₂ O): 1.26 m (2H, -CH ₂ center), 1.51 m (4H, 2CH ₂ ), 2.13 t (2H, 7.16 Hz, CH ₂ CO) 3.25 m (4H, 2NCH ₂ ), 3.72 m (2H, CH ₂ OP), 6.85 d (1H, 9.65 Hz, CH-6 '), 7.97 dd (1H, 2 18, CH-5 '), 8.70 d (1H, CH-3'). ³¹ P NMR (D ₂ O) 2.12 s.

3.2. N- [6-N- (2,4-Dinitrophenyl) aminohexanoyl] -2-aminoethyl ester of triphosphoric acid (Ic)

To a solution of the triethylammonium salt of 2-N- [6-N- (2,4-dinitrophenyl) aminohexanoyl] aminoethyl phosphate (15.4 mg, 25 μmol) in 3 ml abs. DMF was added CDI (32 mg, 0.2 mmol). The reaction mixture was stirred for 18 hours at 4 ° C under argon, then MeOH (0.33 ml, 8 mmol) was added and left for 40 min at 20 ° C. The reaction mixture was concentrated to 2 ml and a 0.3 M solution of (Bu ₃ NH) ₂ H ₂ P ₂ O ₇ (0.5 ml, 150 μmol) was added. The reaction mixture was stirred for 18 hours at 20 ° C, then diluted with water to 10 ml and applied to a Dowex 50 column (2 × 5 cm) in NH ₄ ⁺ form. The substance was eluted with water. The UV absorbing fractions were evaporated, the residue was dissolved in 0.3 ml of water and applied to a LiChroprep RP 8 column (1 × 15 cm) in 5 mM NH ₄ HCO ₃ . Elution was carried out in a linear gradient of MeOH concentrations (0 → 20%) in 5 mM NH ₄ HCO ₃ . Fractions containing the desired product were evaporated, evaporated with water (2 × 5 ml) and lyophilized. Yield 5.5 mg (8.8 μmol, 35%).

UV (water) λmax 363 (ε 17500), λmax 265 (ε 8340).

¹ H NMR (D ₂ O): 1.28 m (2H, -CH ₂ center), 1.46 q (2H, 6.48, -CH ₂ -), 1.55 q (2H, 6.48 , -CH ₂ -), 2.13 t (2H, 7.47 Hz, CH ₂ CO), 3.27 t (1H, 4.98, NH CH ₂ ), 3.34 t (1H, 7.16 , NH CH ₂ ), 3.88 m (2H, CH ₂ OR), 6.97 d (1H, 9.65 Hz, CH-6 '), 8.10 dd (1H, 2.8, CH-5 '), 8.93 d (1H, CH-3').

³¹ P NMR (D ₂ O) -6.38 d (J _{Pα, Pβ} 21.4 P _α ), -10.32 d (J _{Pβ, Pγ} 19.3 P _γ ), -21.77 t (P _β )

Example 4. Obtaining N- (Fluoresceinaminothiocarbonyl) -8-amino-3,6-dioxaoctyl ester of triphosphoric acid (Id).

The synthesis of compound Ig was carried out according to the following scheme:

4.1. N- (9-Fluorenylmethoxycarbonyl) -8-amino-3,6-dioxaoctan-1-ol

Monomethoxytrityl chloride (2.7 g, 8.9 mmol) was added to a solution of triethylene glycol (10 g, 66.5 mmol) in pyridine (50 ml). The reaction mass was kept for 18 h at 37 ° C. The product was extracted with chloroform in the presence of aqueous NaHCO ₃ . The chloroform extracts were evaporated, the residue was dissolved in pyridine (20 ml), cooled to 0 ° C, and methanesulfonyl chloride (1.38 ml, 17.8 mmol) was added. The reaction mass was kept for 18 h at 5 ° С, and CCl _{4 was} extracted in the presence of aqueous NaHCO ₃ . The organic extracts were evaporated, the residue was dissolved in dimethylformamide (30 ml), NaN ₃ (1.16 g, 17.8 mmol) was added. The reaction mass was stirred for 5 hours at 90 ° C, the solvent was removed in vacuo, the residue was extracted with CCl ₄ in the presence of water. The organic extracts were evaporated, the residue was dissolved in dimethylformamide (30 ml), mercaptoethanol (3 ml) and 25% aqueous ammonia (0.5 ml) were added. The reaction mass was held for 18 h at room temperature, applied to a Dowex-50 column (NH ₄ ⁺ form, 3 × 6 cm), the column was washed with methanol (50 ml), then eluted with a mixture of methanol (90 ml) and 25% aqueous ammonia ( 10 ml). The solvents were evaporated, the residue was evaporated with pyridine (2 × 20 ml), dissolved in pyridine (20 ml) and 9-fluorenylmethoxycarbonyl chloride (2.3 g, 8.9 mmol) was added and kept at room temperature for 18 hours. The reaction mass was diluted with aqueous NaHCO ₃ and extracted with CCl ₄ (3 × 20 ml). The organic layers were evaporated, evaporated with toluene (3 × 20 ml), 80% aqueous acetic acid (50 ml) was added. After 18 h at room temperature, the reaction mass was evaporated and chromatographed on a column (2 × 25 cm) with silica gel (63-100 μm), eluted with 3% methanol in chloroform. Yield 440 mg (13%).

UV (H ₂ O) λ _max 265 (ε 17500).

¹ H-NMR (CDCl ₃ + CD ₃ OD): 7.76 d, 7.60 d [4H, H-1, 4, 5, 8 (Fmoc)], 7.39 t, 7.31 t [4H J 7.5, H-2, 3, 6, 7 (Fmoc)], 4.41 d (2H, J 6.9, CH ₂ (Fmoc)), 4.21 t [1H, J 6.9 , H-9 (Fmoc)], 3.70-3.56 m (10H, 5 CH ₂ ), 3.39 m (2H, CH ₂ N).

4.2. N- (9-Fluorenylmethoxycarbonyl) -8-amino-3,6-dioxaoctane ester of triphosphoric acid

A solution of N - [(9-Fluorenylmethoxycarbonyl) -8-amino-3,6-dioxaoctanol (220 mg, 0.59 mmol) in triethyl phosphate (1 ml) was cooled to 0 ° C and phosphorus oxychloride (133 μl, 1.43 was added mmol). The reaction mass was held for 18 h at 5 ° C and a mixture of tributylamine (1 ml, 4.3 mmol) and 0.8 mmol of a solution of bis (tributylammonium) pyrophosphate (3 ml, 2.4 mmol) in dimethylformamide was added with vigorous stirring. The reaction mixture was stirred for 3 hours at room temperature, applied to a Dowex-50 column (NH ₄ ⁺ form, 2 × 4 cm), and the column was washed with 50% aqueous methanol (50 ml). The solvents were evaporated and chromatographed on a column (2 × 18 cm) with LiChroprep RP-18 (25–40 μm) in a gradient of methanol concentration in water (0 → 20%). Yield 40 mg (11%).

UV (H ₂ O) λmax 265 (ε 17000).

¹ H-NMR (D ₂ O): 7.80 d, 7.59 d [4H, J 7.5, H-1, 4, 5, 8 (Fmoc)], 7.41 t, 7.33 t [4H, J 7.5, H-2, 3, 6, 7 (Fmoc)], 4.47 m (2H, CH ₂ (Fmoc)), 4.18 m [1H, H-9 (Fmoc)] 4.02 m (2H, CH ₂ OP), 3.64-3.38 m (8H, 4 CH ₂ ), 3.11 m (2H, CH ₂ N). ³¹ P-NMR (D ₂ O): -9.76 d (1P, J 19.3, P _γ ), -10.49 d (IP, J 19.3, Pα), -22.49 t (1P , P _β ).

4.3. N- (Fluoresceinaminothiocarbonyl) -8-amino-3,6-dioxaoctyl ester of triphosphoric acid

Dissolved N - [(9-Fluorenylmethoxycarbonyl) -8-amino-3,6-dioxo-octane ester of triphosphoric acid (20 mg, 0.03 mmol) in 25% aqueous ammonia (5 ml). After 16 hours at 5 ° C, the reaction mass was filtered, the filtrate was evaporated to a volume of about 1 ml, dimethylformamide (1 ml) was added, fluorescein isothiocyanate (20 mg, 0.05 mmol) and triethylamine (20 μl) were added. The reaction mass was stirred for 18 hours at room temperature, evaporated, evaporated with water (3 × 10 ml) and applied to a column (2 × 18 cm) with LiChroprep RP-8 (25-40 μm). Eluted with 0.05 M aqueous NH ₄ HCO ₃ . Yield 1 mg (5%).

UV (H ₂ O, pH 9.0) λmax 492 (ε 65000).

¹ H-NMR (D ₂ O): 6.94 m, 7.50 m, 7.76 m, 7.92 s (9H, fluorescein), 4.25 m (2H CH ₂ OP), 3.89 m (10H, 5 CH ₂ ). ³¹ P-NMR (D ₂ O): -9.84 d (1P, J 18.3, P ^γ ), -10.49 d (1P, J 19.3, P _α ), - 22.59 dd ( 1P, P _β ).

Example 5. Obtaining N- (Rhodaminaminothiocarbonyl) -2-aminoethyl ester of triphosphoric acid (Ie).

The synthesis of compound Id was carried out according to the following scheme:

where RdB is a radical of the formula:

5.1. N- (Rhodaminaminothiocarbonyl) -2-aminoethyl ester of phosphoric acid

Phosphoric acid aminoethyl ester (141 mg, 1 mmol) was dissolved in dimethylformamide (5 ml), rhodamine B was added to isothiocyanate (107 mg, 0.2 mmol), methylimidazole (40 μl) and triethylamine (200 μl). The reaction mixture was stirred for 18 h at room temperature, evaporated, dissolved in water (20 ml), the solution was extracted with CCl ₄ (3 ml), n butanol (2 × 3 ml) and applied to a column (2.2 × 12 cm) s DEAE cellulose was eluted with a 30% MeOH solution in a concentration gradient of NH ₄ HCO ₃ (0 → 0.2 M). The desired fractions were combined and lyophilized. Yield 40 mg (20%).

UV (H ₂ O) λ _max 556 (ε 59000). ¹ H-NMR (D ₂ O): 8.09 m, 7.74 m, 7.61 m, 7.22 m, 7.07 m, 6.97 m, 6.62 m, 6.36 s, 6 28 s (9H, PdB), 3.89 m (2H, CH ₂ O), 3.69 m (2H, CH ₂ N), 3.31 m (8H, 4 × CH ₂ CH ₃ ), 0, 99 m (12H, 4 × CH ₂ CH ₃ ). ³¹ P-NMR (D ₂ O): 2.59 s

5.2. N- (Rhodaminaminothiocarbonyl) -2-amino-ethyl ester of triphosphoric acid

Phosphoric acid N- (Rhodaminaminothiocarbonyl) -2-aminoethyl ester (40 mg, 0.045 mmol) was dissolved in dimethylformamide (3 ml), two times were evaporated with dimethylformamide (2 × 3 ml), CDI (73 mg, 0.45 mmol) was added. . The reaction mixture was stirred for 18 h at 4 ° С in argon atmosphere, then MeOH (0.33 ml, 8 mmol) was added and left for 40 min at 20 ° С. The reaction mixture was concentrated to 2 ml and 0.45 M (Bu ₃ NH) ₂ H ₂ P ₂ O ₇ in DMF (1.5 ml) was added. The reaction mixture was stirred for 18 h at 20 ° C, diluted with water (up to 20 ml), the solution was extracted with CCl ₄ (3 ml), n butanol (2 × 3 ml) and applied to a column (2.5 × 10.5 cm) with DEAE cellulose, was eluted with a 30% MeOH solution in a concentration gradient of NH ₄ HCO ₃ (0 → 0.3 M). The desired fractions were combined, concentrated in vacuo and the residue was lyophilized. Yield 9.7 mg (27%).

UV (H ₂ O) λ _max 556 (ε 59000). ¹ H-NMR (D ₂ O): 8.26 m, 7.77 m, 7.49 m, 7.41 m, 7.13 m, 6.71 m, 6.51 s, 6.40 s ( 9H, PdB), 4.13 m (2H, CH ₂ O), 3.81 m (2H, CH ₂ N), 3.41 m (8H, 4 × CH ₂ CH ₃ ), 1.1 m (12H , 4 × CH ₂ CH ₃ ). ³¹ P-NMR (D ₂ O): -9.3 d (1P, J 18.3, P _γ ), -10.47 d (1P, J 17.3, P _α ), -22.24 q ( 1P, J 18.3, P _β ).

Example 6. Obtaining N- (Rhodaminaminothiocarbonyl) -4-aminobutyl ester of triphosphoric acid (Ie).

The synthesis of compound Ie was carried out as follows:

6.1. N- (9-Fluorenylmethoxycarbonyl) -4-aminobutanol

A solution of Na ₂ CO ₃ (860 mg, 8 mmol) in water (8 ml) was added to aminobutanol (186 μl, 2 mmol), a solution of 9-fluorenylmethoxycarbonyl chloride (400 mg, 1.6 mmol) in dioxane (800 μl) was added and kept for 18 hours at room temperature. The desired product precipitated, it was filtered off and washed with water. Yield 446 mg (93%).

UV (H ₂ O) λ _max 265 (ε 17500).

¹ H-NMR (D ₂ O): 7.76 d, 7.60 d [4H, H-1, 4, 5, 8 (Fmoc)], 7.39 t, 7.31 t [4H, J 7 5, H-2, 3, 6, 7 (Fmoc)], 4.41 d (2H, J 6.9, CH ₂ (Fmoc)), 4.21 t [1H, J 6.9, H- 9 (Fmoc)], 3.77 m (2H, CH ₂ N), 2.84 m (2H, CH ₂ O), 1.35-1.23 m (4H, 2-CH ₂ -).

6.2. N- (9-Fluorenylmethoxycarbonyl) -4-aminobutyl ester of triphosphoric acid

A solution of (9-fluorenylmethoxycarbonyl) aminobutanol (220 mg, 0.59 mmol) in triethyl phosphate (1 ml) was cooled to 0 ° C and phosphorus oxychloride (133 μl, 1.43 mmol) was added. The reaction mixture was held for 18 h at 5 ° C and a mixture of tributylamine (1 ml, 4.3 mmol) and a 0.8 mM solution of bis (tributylammonium) pyrophosphate (3 ml, 2.4 mmol) in dimethylformamide was added with vigorous stirring. The reaction mixture was stirred for 3 hours at room temperature, applied to a Dowex-50 column (NH ₄ ⁺ form, 2 × 4 cm), eluted with 50% aqueous methanol (50 ml). Solvents were removed in a vacuum of a water-jet pump, the residue was chromatographed on a column (2 × 18 cm) with LiChroprep RP-18 (25–40 μm) in a methanol concentration gradient in water (0 → 20%). Yield 40 mg (11%).

UV (H ₂ O) λ _max 265 (ε 17500).

¹ H-NMR (D ₂ O): 7.74 d, 7.50 d [4H, J 7.5, H-1, 4, 5, 8 (Fmoc)], 7.33 t, 7.25 t [4H, J 7.5, H-2, 3, 6, 7 (Fmoc)], 4.39 d (2H, J 6.9, CH ₂ (Fmoc)), 4.10 t [1H, J 6 9, H-9 (Fmoc)], 3.77 m (2H, CH ₂ O), 2.84 m (2H, CH ₂ N), 1.35-1.23 m (4H, 2-CH ₂ -). ³¹ P-NMR (D ₂ O): -10.34 d (1P, J 18.3, P _γ ), -10.44 d (1P, J 17.3, P _α ), -22.79 q ( 1P, J 18.3, P _β ).

6.3. N- (Rhodaminaminothiocarbonyl) -4-aminobutyl ester of triphosphoric acid 1e.

Dissolved N - [(9-Fluorenylmethoxycarbonyl) -4-aminobutyl ester of triphosphoric acid (22 mg, 0.04 mmol) in 25% aqueous ammonia (5 ml). After 16 hours at 5 ° C, the reaction mass was filtered, the filtrate was evaporated to a volume of about 1 ml, dimethylformamide (5 ml) and triethylamine (0.5 ml) were added, evaporated to a volume of about 2 ml, rhodamine B was added to isothiocyanate (43 mg, 0 , 08 mmol), methylimidazole (40 μl) and triethylamine (80 μl). The reaction mixture was stirred for 18 h at room temperature, evaporated, dissolved in water (20 ml), the solution was extracted with CCl ₄ (3 ml), n butanol (2 × 3 ml) and applied to a column (2.5 × 10.5 cm ) with DEAE cellulose, was eluted with a 30% MeOH solution in a concentration gradient of NH ₄ HCO ₃ (0 → 0.2 M). The desired fractions were combined, concentrated in vacuo and the residue was lyophilized. Yield 25 mg (75%).

UV (H ₂ O) λ _max 556 (ε 59000).

¹ H-NMR (D ₂ O): 7.75 m, 6.87 m, 6.54 m, 6.38 m, 6.25 m (9H, PdB), 3.87 m (2H, CH ₂ O) ), 3.21 m (10H, CH ₂ N and 4 × CH ₂ CH ₃ ), 1.53 m (4H, 2 × -CH ₂ -), 0.91 m (12H, 4 × CH ₂ CH ₃ ) . ³¹ P-NMR (D ₂ O): -10.07 d (IP, J 18.3, P _γ ), -10.34 d (IP, J 17.3, P _α ), -21.94 q ( 1P, J 18.3, P _β ).

Example 7. N- (Fluoresceinaminothiocarbonyl) -4-aminobutyl ester of triphosphoric acid (1g).

The synthesis of compound 1g was carried out according to the following scheme:

Triphosphoric acid N- (Fluoresceinaminothiocarbonyl) -4-aminobutyl ester (1g) was obtained from triphosphoric acid 4-aminobutyl ester according to the method described in Example 6 for the synthesis of N- (rhodaminaminothiocarbonyl) -4-aminobutyl ester of triphosphoric acid (1eocyanate) . Yield 21 mg (71%).

UV - Visible: (H ₂ O, pH 9.0) λ _max 493 nm (ε 65000). ¹ H-NMR (D ₂ O): 6.94 m, 7.50 m, 7.76 m, 7.92 s (9H, fluorescein), 3.81 m (2H, CH ₂ O), 2.95 m (2H, CH ₂ N), 1.61-1.52 m (4H, 2-CH ₂ -). ¹³ P-NMR (D ₂ O): -10.22 d (IP, J 18.1, P _γ ), -10.58 d (IP, J 17.1, P _α ), -22.13 dd ( IP, P _β ).

Example 8. N- (Eosinaminothiocarbonyl) -4-aminobutyl ester of triphosphoric acid (1h).

The synthesis of compound 1z was carried out according to the following scheme:

N - [(Zosinaminothiocarbonyl) -4-aminobutyl ester of triphosphoric acid (1h) was obtained from 4-aminobutyl ester of triphosphoric acid according to the method described in Example 6 for the synthesis of N- (rhodaminaminothiocarbonyl) -4-aminobutyl ester of triphosphoric acid (1e) using eosinisothiocyanate. Yield 17 mg (53%).

UV - Visible: (H ₂ O) λ _max 527 nm (ε 65800). ¹ H-NMR (D ₂ O); 8.94 d, (1H, J 7.5), 7.55 d, (1H, J 8.1), 7.1 s, (1H), 7.04 s (2H) (eosin), 3, 91 m (2H, CH ₂ O), 2.95 m (2H, CH ₂ N), 1.78-1.67 m (4H, 2 × -CH ₂ -). ¹³ P-NMR (D ₂ O): -10.12 d (IP, J 18.2, P _γ ), -10.48 d (IP, J 17.5, P _α ), -22.14 dd ( IP, P _β ).

Example 9. N- (Tetramethylrodaminaminothiocar6onyl) -4-aminobutyl ester of triphosphoric acid (1i).

The synthesis of compound 1i was carried out according to the following scheme:

Triphosphoric acid N- (tetramethylrodaminaminothiocarbonyl) -4-aminobutyl ester (1i) was prepared from triphosphoric acid 4-aminobutyl ester according to the method described in Example 6 for the synthesis of N- (rhodaminaminothiocarbonyl) -4-aminobutyl triphosphoric acid tetraethosphate (1-tetramethoxy tetraethosulfonate) (1) . Yield 27 mg (65%).

UV - Visible: (H ₂ O) λ _max 552 nm (ε 59300). ¹ H-NMR (D ₂ O): 7.75 m, 6.87 m, 6.54 m, 6.38 m, 6.25 m (9H, tetramethylrodamine), 3.85 m (2H, CH ₂ O ), 3.02 m (2H, CH ₂ N), 1.53 m (4H, 2x-CH ₂ -), 1.91 m (12H, 4 × -CH ₃ ). ¹³ P-NMR (D ₂ O): -10.17 d (IP, J 18.1, P _γ ), -10.42 d (IP, J 17.6, P _α ), -22.09 dd ( IP, P _β ).

Example 10. N - [(6-N-Biotinylaminohexanoyl)] - 4-aminobutyl ester of triphosphoric acid (1 k).

The synthesis of compound 1k was carried out according to the following scheme:

N - [(6-N-biotinylaminohexanoyl)] - triphosphoric acid 4-aminobutyl ester (1 k) was obtained from triphosphoric acid 4-aminobutyl ester according to the method described in Example 6 for the synthesis of tri- phosphoric N- (rhodaminaminothiocarbonyl) -4-aminobutyl ester acid (1e) using 6-N-biotinylaminohexanoic acid N-oxysuccinimide ester. After the reaction and extraction, the residue was diluted with water to 20 ml and applied to a column (2.5 × 4.5 cm) with DEAE cellulose (HCO ₃ ^- ). The column was washed with 30% MeOH (50 ml), 0.12 M NH ₄ HCO ₃ in 30% MeOH (100 ml), compound (1k) was eluted with a 30% solution of MeOH in 0.25 M NH ₄ HCO ₃ (100 ml), concentrated in vacuo, the residue was dissolved in water and lyophilized. Yield 13 mg (47%).

UV (H ₂ O) λ _max 195 nm (ε 2220). ¹ H-NMR (D ₂ O): 4.25 m and 4.05 m (2H, 2 × CH NH (Bio)), 3.85 m (2H, CH ₂ O), 3.15 m (1H, CHS (Bio), 3.02 m (2H, CH ₂ N), 2.83 d (1H, J 13.5, SCH _a ), 2.61 d, (1H, J 13.5, SCH _b ), 2.17 t + 2.23 t (4H, J 7 each, 2 × CH ₂ CO), 1.68-1.28 m (14H, 7 × -CH ₂ -). ¹³ P-NMR (D ₂ O ): -9.72 d (IP, J 19.1, P _γ ), -10.42 d (IP, J 20.1, P _α ), -21.9 dd (1P, P _β ).

Example 11. 4- {3 - [(1,3,5,7-Tetramethyl-4,4-difluoro-3a, 4a-diaza-4-boraindacen-2-yl) propionyl]} aminobutyl ester of triphosphoric acid (1 l) .

The synthesis of compound 1l was carried out according to the following scheme:

4- {3 - [(1,3,5,7-Tetramethyl 4,4-difluoro-3a, 4a-diaza-4-boraindacen-2-yl) propionyl]} aminobutyl ester of triphosphoric acid (1k) was obtained from 4- aminobutyl ester of triphosphoric acid according to the method described in example 6 for the synthesis of M- (rhodaminaminothiocarbonyl) -4-aminobutyl ester of triphosphoric acid (1e) using 3- (1,3,5,7-tetramethyl-4,4-N-oxysuccinimide ester difluoro-3a, 4a-diaza-4-boraindacen-2-yl) propionic acid. Yield 17 mg (47%).

UV - Visible: (H ₂ O) λ _max 512 nm (ε 72000). ¹ H-NMR (D ₂ O): 7.35 s (1H, H-8), 6.14 s (1H, H-6), 3.85 m (2H, CH ₂ O), 2.89 m (2H, CH ₂ N), 2.72 t (2H, J 8.0, - CH ₂ CH ₂ CO), 2.49 t (2H, -CH ₂ CH ₂ CO), 2.45 s and 2, 44 s (6H, 3-CH ₃ and 5-CH ₃ ), 2, 23 s and 2, 25 s (6H, 1-CH ₃ and 7-CH ₃ ), 1.68-1.58 m (4H, 2 × -CH ₂ -). ¹³ P-NMR (D ₂ O): -10.05 d (IP, J 18.3, P _γ ), -10.38 d (IP, J 17.8, P _α ), -22.21 dd ( IP, P _β ).

Example 12. Determination of the substrate activity of new triphosphates of the formula I.

The substrate activity was determined according to the method described previously [Dyatkina IB, von Yant-Lipinsky M., Minasyan Sh.Kh., Kukhanova MK, Kraevsky AA, Chidzhavadze Z.G., Bibilashvili Z.Sh ., Bioorgan. Chemistry, 1987, 13, No. 10, 1366-1374], the essence of which is as follows. In the work, oligonucleotides of different composition and length were used. The nucleotide sequence of some oligonucleotides is shown below:

5'- ³² P-dCCC AGT CAC GAC GT

5'- ³² P-dCCG TSA ATT SST GTA GTC

5'- ³² P-dCCG TSA ATT SST GTA GTC TCG

5'- ³² P oligonucleotides were obtained using [γ- ³² P] -ATP and T4 polynucleotide kinase. The reaction mixture contained (in a volume of 20 μl) 20 pmol of oligonucleotide, 50 μCi [γ- ³² P] -ATP, 2 units. enzyme and buffer. The reaction mixture was incubated for 30 min at 37 ° C, stopped by heating for 15 min at 65 °. The primer oligonucleotide was purified on a 1.5 M BioGel A column and used to introduce various labels at the 3 ′ end. The reaction mixture in a volume of 10 μl contained 0.02 μm 5 '- [ ³² P] -oligonucleotide, 0.2 units Act. TDT, 100 mM sodium cacodylate (pH 7.2), 2 mM CoCl ₂ , 0.1 mM dithiothreitol and substrates in various concentrations. The reaction mixture was incubated for 10 min at 37 ° C. The reaction was stopped by the addition of 5 μl of formamide containing 0.5 mM EDTA and 0.1% phenol blue and xylencyanol. The products were separated in a 15% denaturing polyacrylamide gel. The gels were exposed with a Kodak RX X-ray film. The results are presented in figures 1, 2 and in the table.

Figure 1 shows the separation of the reaction products in a denaturing 15% polyacrylamide gel after incorporation of compounds 1a and 1b into the 3'-end of oligonucleotide (1) using TDT: track 1 — position of the primer oligonucleotide; tracks 2-4 — inclusion of ddATP used as a control at concentrations of 0.01, 0.1, and 1 μM, respectively; tracks 5-10 inclusion 1a at concentrations of 0.05, 0.1, 0.25, 0.5, 1 and 2 μM, respectively; tracks 11-16 - inclusion of 1b at concentrations of 0.05, 0.1, 0.25, 0.5, 1 and 2 μM, respectively.

Figure 2 illustrates the separation of the reaction products in a denaturing 15% polyacrylamide gel after incorporation of compound 1c into the 3'-end of the terminal deoxynucleotide transferase oligonucleotide: tracks 1-3 - ddATP incorporation at concentrations of 0.02, 0.05 and 0.5 μM, respectively ; tracks 4–7 — inclusion of 1c at concentrations of 0.2, 1, and 10 μM, respectively.

As can be seen from the drawings and table data, 50% utilization of the oligonucleotide occurs already at concentrations of synthesized triphosphates of 0.2-8 μM, which allows us to consider the new compounds as effective TDT substrates.

Table Compound R X Concentration resulting in 50% utilization of μM oligonucleotide 1a Dansil NH (CH ₂ ) ₄ 1-5 1b Dansil NH (CH ₂ ) ₅ C (O) NH (CH ₂ ) ₂ 4-7 1c Dinitrophenyl NH (CH ₂ ) ₅ C (O) NH (CH ₂ ) ₂ 5-8 1r Fluorescein NHC (S) NHCH ₂ CH ₂ [O (CH ₂ ) ₂ ] ₂ 0.2-0.8 1d Rhodamine B NH (CH ₂ ) ₂ 1-3 1e Rhodamine B NH (CH ₂ ) ₄ 0.6-1 1g Fluorescein NH (CH ₂ ) ₄ 1-5 1z Eosin NH (CH ₂ ) ₄ 2-5 1i Tetramethylrodamine NH (CH ₂ ) ₄ 0.7-1 1 to Biotin NH (CH ₂ ) ₅ C (O) NH (CH ₂ ) ₄ 0.3-0.8 1l Bodipy NH (CH ₂ ) ₄ 2-8

Claims (1)

New substrates of terminal deoxynucleotidyl transferase of general formula I

where R is a marker group selected from dansil, dinitrophenyl, fluorescein, fluorenylmethyloxycarbonyl, rhodamine B, tetramethylrodamine, eosin, erythrosine, trioxalene, BODIPY, biotin, auramine, psoralen, pyrene, Texas red, actinomycicin D, 7; X is a linker, which is a linear carbon chain that contains heteroatoms, such as nitrogen and oxygen, and the chain may contain carbonyl and thiocarbonyl as functional groups.

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