RU2415842C1 - Etidium ethylsulphate - fluorescent dye for detecting nucleic acids - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: present invention relates to organic chemistry and specifically to a novel chemical compound - fluorescent dye of etidium ethylsulphate (3,8-diamino-5-ethyl-6- phenylphenantridium ethyl sulphate) of formula I

which increases fluorescence intensity when exposed to UV light as a result of bonding with nucleic acids.

EFFECT: obtaining a novel compound which can be used to detect DNA.

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Description

The invention relates to a new chemical compound, specifically to ethidium ethyl sulfate (3,8-diamino-5-ethyl-6-phenylphenanthridine ethyl sulfate), of the formula I:

which can be used as a fluorescent dye to detect nucleic acids.

An analog of ethidium ethyl sulfate - ethidium bromide is widely used in molecular biology, genetic engineering and diagnostic medicine for the detection of nucleic acids {[1]. Maniatis T., Fritsch E., Sambrook J. Methods of genetic engineering. Molecular Cloning Per. from English M .: Mir, 1984. 480 p., [2]. Pat. 4683202 U.S., Int. C1. C12Q 1/68, C12N 15/10, B01L 7/00, C07K 14/795, C07K 14/805. US C1. 435 / 91.2, 435 / 317.1, 435 / 320.1, 536 / 23.1, 536 / 24.33. Process for amplifying nucleic acid sequences. [3]. Using molecular marker technology in studies on plant genetic diversity, DNA-based technologies. PCR-based technologies. Sequence-tagged sites (Microsatellites, SCARs, CAPS, ISSRs), IPGRI and Cornell University, 2003. 38 pp.}.

The aim of the invention is the synthesis of a fluorescent dye having the property of enhancing the fluorescence intensity upon exposure to UV light as a result of binding to nucleic acids.

This goal is achieved by a new chemical compound of ethidium with ethyl sulfate of formula (I), which has the property of enhancing the fluorescence intensity upon exposure to UV light as a result of binding to nucleic acids.

The synthesis of compound (I) was carried out in accordance with scheme 1.

The first stage of this synthesis is the quaternization of 3,8-dicarboethoxyamino-6-phenylphenanthridine (II) with diethyl sulfate. Moreover, diethyl sulfate acts as a reagent and solvent. A mixture of 3,8-dicarbethoxyamino-6-phenylphenanthridine (II) and diethyl sulfate was heated for 30 minutes at 160-165 ° C. After cooling, the mixture was triturated with dry ether, the product was filtered, washed with dry ether and dry acetone. Received in 87% yield of ethyl sulfate (III). Strict proof of the composition and structure of the obtained quaternization product is based on the data of elemental analysis and spectrum analysis ( ¹ H NMR, IR and ESP) of the product. The formation of 3,8-dicarboethoxyamino-6-phenylphenanthridinium ethyl sulfate (III) was established in 87% yield.

In the second stage of the synthesis, crude ethyl sulfate (III) was hydrolyzed in a medium of 78% sulfuric acid at 135-140 ° C for 1 hour with stirring to remove carboethoxy groups. The mixture was cooled and poured into ice water. With stirring, a 25% ammonia solution was added dropwise. The mixture was extracted with n. butanol. From n. butanol extract, left at 0 ÷ / + 5 ° C for 16 hours, a fine crystalline dark red precipitate was filtered off, dried in air and recrystallized from hot water. Received in 61% yield of ethidium ethyl sulfate (I).

The structure of compound (I) was established on the basis of elemental analysis, ¹ H NMR spectrum, which is similar to the spectrum of ethidium bromide {[4]. Firth WJ, Watkins CL, Graves DE, Yielding LW // J. Heterocycl. Chem. 1983. V.20. N 3. P.759-765}, and IR and ESP spectra. The fluorescence emission spectra of aqueous solutions of these two compounds are almost identical.

A comparative description of the practical application of the new fluorescent dye of ethidium ethyl sulfate (I) and the standard ethidium bromide used for DNA detection was performed. The sensitivity of these dyes was almost the same when detecting amplified DNA obtained by polymerase chain reaction. Thus, the new compound can be used to replace ethidium bromide in detecting nucleic acids.

IR spectra were recorded on a Vector 22 spectrometer in KBr pellets, electronic absorption spectra (EES) on a Specord UV-vis spectrophotometer (10 ^-4 N solutions in alcohol), fluorescence emission spectra (SEF) on a Cara Eclipse fluorescence spectrophotometer from the company “Varian” (excitation at wavelengths having λ _max : 247, 333 and 486 nm, irradiation of 10 ^-4 N solutions in H ₂ O). Melting points were determined on a Kofler apparatus. ¹ H NMR spectra were recorded on a Bruker AC 200 instrument ( ¹ H - 200.13 MHz) for solutions at 25 ° C with resonance stabilization by the deuterium signal of the DMSO-D ₆ solvent. Chemical shifts (ppm) were measured relative to signals of internal standards: DMSO (δ _n 2.50 ppm).

Figure 1 - Gel staining: A - ethidium ethyl sulfate (0.5 μg / ml), B - ethidium bromide (0.5 μg / ml).

Figure 2 - Gel staining: A - ethidium ethyl sulfate (0.5 μg / ml), B - ethidium bromide (0.5 μg / ml). The last lane is the products of specific DNA hydrolysis of plasmid pBluescript IIKS (-) with the restriction endonuclease MspI.

The invention is illustrated by examples.

Example 1. 3,8-Dicarboethoxyamino-5-ethyl-6-phenylphenanthridinium ethyl sulfate (III). A mixture of 1.48 g (3.45 mmol) of 3,8-dicarboethoxyamino-6-phenylphenanthridine (II) and 2.59 g (16.8 mmol) of diethyl sulfate was heated for 30 min at 160-165 ° C. Intensive crystallization was observed in the initially formed solution. After cooling, the mixture was triturated with 10 ml of dry ether, the product was filtered, washed with 3 × 3 ml of dry ether, 3 × 3 ml of dry acetone. Received 1.74 g (87%) of ethyl sulfate (III), so decomp. 200-220 ° C (without melting).

Found,%: C 59.47, 59.32; H 5.97, 5.87; N, 7.31; 7.32; S 5.50, 5.70. C ₂₉ H ₃₃ N ₃ O ₈ S.

Calculated,%: C 59.67, H 5.71, N 7.20, S 5.49.

¹ H NMR spectrum (5% solution in DMSO-D ₆ , δ / ppm, J / Hz): 1.09, 1.18 and 1.30 (all t, 3 H each, J = 7, three CH ₃ groups, three fragments of C ₂ H ₅ O); 1.51 ears t (3 H, J = 7, CH ₃ group, C ₂ H ₅ N fragment); 3.73, 4.06 and 4.22 (all k, 2 H each, J = 7, three CH ₂ groups _of three C ₂ H ₅ O fragments); 4.63 ears q (2 H, J = 7, CH ₂ group, C ₂ H ₅ N fragment); 7.7-7.9 m (6 H, H-4 or H-7 and C ₆ H ₅ ); 8.02 and 8.18 (both dd, 1 H each, J = 9 and J = 2, H-2, H-9); 8.72 d (1 H, J = 2, H-7 or H-4); 8.95 and 9.01 (both d, 1 H each, J = 9, H-1, H-10); 10.24 and 10.51 (both s, 1 H, 2 NH groups).

IR spectrum, KBr: 720, 730, 760, 780, 930, 1030, 1080, 1090, 1100, 1160, 1190, 1240, 1320, 1350, 1380, 1410, 1490, 1540, 1570, 1590, 1610, 1630, 1730 cm ^-1 (C = O). ESP (EtOH) λ _max / nm (log ε): 217 (4.54), 285 (4.80) and 425 (3.82).

Example 2. 3,8-Diamino-5-ethyl-6-phenylphenanthridinium ethyl sulfate (ethidium ethyl sulfate) (I). A solution of 1.00 g (1.74 mmol) of ethyl sulfate (III) in 2.79 ml of 78% sulfuric acid (containing 37.8 mmol of H ₂ SO ₄ ) was heated for 1 h at 135-140 ° C with stirring. In the resulting dark-colored solution, gas evolution was observed, at the end of the exposure, the solution brightened, gas evolution was noticeably weaker. The mixture was cooled and poured into 2.5 ml of ice water. With stirring, 10 ml of a 25% solution of ammonia (133 mmol NH ₃ ) was added dropwise, and the formation of a dark red gummy precipitate was observed. The mixture was extracted 2 times with 4 ml of N. butanol. The aqueous layer after extraction became almost colorless. From n. butanol extract, left at 0 ÷ / + 5 ° C for 16 hours, a fine crystalline dark red precipitate was filtered off, dried in air and recrystallized from 5.2 ml of hot water. Received 0.48 g (61%) of ethidium ethyl sulfate (I), so pl. 250-252 ° C (decomp.).

Found,%: C 63.23, 62.90; H 5.89, 5.87; N, 9.68; 9.65; S 7.60, 7.30. C ₂₃ H ₂₅ N ₃ O ₄ S.

Calculated,%: C 62.84, H 5.74, N 9.56, S 7.29.

¹ H NMR spectrum (5% solution in DMSO-D ₆ , δ / ppm, J / Hz): 1.10 t (3 H, J = 7, CH ₃ ethyl sulfate group); 1.40 ears t (3 H, J = 7, CH ₃ group, C ₂ H ₅ N fragment); 3.74 k (2 H, J = 7, CH ₂ ethyl sulphate group); 4.47 ears q (2 H, J = 7, CH ₂ group, C ₂ H ₅ N fragment); 5.93 and 6.36 (both ears. With 2 H, 2 groups of NH ₂ ); 6.25 d (1 H, J = 2, H-7); 7.3-7.4 m (2 H, H-2, H-4); 7.51 dd (1 H, J = 9 and J = 2, H-9); 7.7-7.8 m (5 H, C ₆ H ₅ ); 8.60 and 8.65 (both d, 1 H each, J = 9, H-10 and H-1, respectively). The assignment of signals in the ¹ H NMR spectrum was made by analogy with the above lit. data [4] for ethidium bromide.

IR spectrum, KBr: 720, 790, 840, 930, 1030, 1070, 1170, 1190, 1220, 1240, 1280, 1330, 1410, 1480, 1490, 1510, 1640 cm ^-1 . ESP (MeOH), λ _max / nm (log ε): 216 (4.61), 293 (4.81) and 528 (3.81). SEF (H ₂ O): λ _max 619 nm. For ethidium bromide company "Fluka" SEF (H ₂ O): λ _max 619 nm.

Example 3. Comparison of the sensitivity of detection of double-stranded DNA with ethidium bromide and ethidium ethyl sulfate.

To test the sensitivity of detection of double-stranded DNA with ethidium bromide and ethidium ethyl sulfate, the products of specific hydrolysis of the DNA of the plasmid pBluescript IIKS (-) with the restriction endonuclease MspI in amounts of 200 ng, 150 ng, 100 ng and 50 ng per lane were analyzed by electrophoresis in 6% acrylamide gel. To obtain a 6% or 8% PAAG, 1/100 PSA and 1/1000 TEMED were added to a 6% or 8% solution of PAA in 0.5 × TBE. The gel was poured between two glasses arranged horizontally and connected by clamps, a comb was inserted. After polymerization, the gel was used for electrophoresis.

Vertical electrophoresis was performed in 0.5 × TBE buffer. The solution of the analyzed DNA was mixed with application buffer (1:10) and placed in the pockets of the gel, subjected to electrophoresis (10 V / cm). The gel was stained with ethidium bromide or ethidium ethyl sulfate (0.5 μg / ml) for 15 min, DNA was visualized under UV light. The image was captured using a Watec AD-901 CD camera (Watec Co., LTD. Japan). The comparison results are presented in figure 1.

From the drawing it can be seen that both methods of staining the gel have almost the same sensitivity.

Example 4. The use of ethidium bromide and ethidium ethyl sulfate to detect PCR products after electrophoresis in 5% polyacrylamide gel.

The amplification and analysis of the exact tandem repeat of ETRD on DNA samples of 60 M. tuberculosis isolates from the West Siberian region of the Russian Federation were used for verification. PCR was performed with oligonucleotide primers

ETRD-1 GTCAAACAGGTCACAACGAGAGGAA

ETRD-2 CCTCCACAATCAACACACTGGTCAT

in a final volume of 20 μl containing 67 mm Tris. - HCl (pH 8.9), 16 mm ammonium sulfate; 1.5 mM MgCl ₂ ; 0.01% Tween 20; 0.2 mM dNTP; 0.5 μM solutions of oligonucleotide primers, 1 unit Act. Taq DNA polymerase.

The reaction was carried out on a Tertsik thermocycler (DNA-Technology, Russia) with initial denaturation at 96 ° C for 2 min, then for 35 cycles with denaturation at 95 ° C for 5 sec, annealing at 62 ° C for 10 sec and elongation at 72 ° C for 10 sec. Final elongation was carried out at 72 ° C for 5 min. The amplification product was analyzed on a 6% acrylamide gel.

The results of the detection of PCR products are presented in figure 2.

Analysis of the obtained electrophoregrams allows us to conclude that ethidium sulfate is suitable for detecting PCR products during their electrophoretic analysis.

Moreover, the claimed compound is cheaper compared to ethidium bromide, since its synthesis eliminates the need for reagents and time to replace the intermediate anion with a bromine anion and the isolation of ethidium bromide.

Claims (1)

Ethidium ethyl sulfate of the formula (I),

possessing the properties of a fluorescent dye for detecting nucleic acids.

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