Intercalating fluorescent dyes for labelling nucleic acids and preparation method thereof

Abstract

The novel intercalating fluorescent compounds of exemplary embodiments of the present invention for analyzing nucleic acids, etc. have excellent intercalating efficiency with nucleic acids such as DNA and RNA of biomaterials, and may not only continuously maintain fluorescence properties and efficiency, but also have excellent effects even in terms of storage stability such as temperature and moisture, etc. and biosafety. In addition, the fluorescent compounds have various advantages capable of being dissolved in distilled water, which is a solvent harmless to the human body, and being applied to a wide range of analysis without being limited to the analysis of specific cells and living tissues.

Claims

1. An intercalating dye compound for analyzing biomaterials, wherein the compound is a compound represented by the following Chemical Formula 1 or a salt thereof; ##STR00022## Wherein, X is oxygen or sulfur, and R is —SO.sub.2CH(CH.sub.2), —CH.sub.2N(CH.sub.3).sub.2, or unsubstituted alkyl having 5 to 10 carbon atoms.

2. The compound of claim 1, wherein the compound of Chemical Formula 1 is selected from the group consisting of the following Compounds 8, 9, and 10 ##STR00023##

3. The compound of claim 1, wherein the compound of Chemical Formula 1 is selected from the group consisting of the following Compounds 15, 16, and 17 ##STR00024##

4. The compound of claim 1, wherein the biomaterials are selected from the group consisting of proteins, glycoproteins, siRNA, DNA and RNA.

5. The compound of claim 1, wherein the compound is intercalated between the biomaterials.

6. A contrast agent composition for labeling biomaterials comprising a compound represented by the following Chemical Formula 1 or a salt thereof: ##STR00025## Wherein, X is oxygen or sulfur, and R is —SO.sub.2CH(CH.sub.2), —CH.sub.2N(CH.sub.3).sub.2, or unsubstituted alkyl having 5 to 10 carbon atoms.

7. The contrast agent composition for labeling the biomaterials of claim 6, wherein the compound of Chemical Formula 1 is selected from the group consisting of the following Compounds 8, 9, and 10 ##STR00026##

8. The contrast agent composition for labeling the biomaterials of claim 6, wherein the compound of Chemical Formula 1 is selected from the group consisting of the following Compounds 15, 16, and 17 ##STR00027##

9. The contrast agent composition for labeling the biomaterials of claim 6, wherein the biomaterials are selected from the group consisting of proteins, glycoproteins, siRNA, DNA and RNA.

10. The contrast agent composition for labeling the biomaterials of claim 6, wherein the compound is intercalated between the biomaterials.

11. A kit for detecting biomaterials formed by including a contrast agent composition consisting of a compound represented by the following Chemical Formula 1 or a salt thereof: ##STR00028## Wherein, X is oxygen or sulfur, and R is —SO.sub.2CH(CH.sub.2), —CH.sub.2N(CH.sub.3).sub.2, or unsubstituted alkyl having 5 to 10 carbon atoms.

12. The kit for detecting the biomaterials of claim 11, wherein the biomaterials are selected from the group consisting of proteins, glycoproteins, siRNA, DNA and RNA.

13. The kit for detecting the biomaterials of claim 11, wherein the compound is intercalated between the biomaterials.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The above and other aspects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

(2) FIG. 1 illustrates a fluorescence intensity shown when DNA is synthesized using a fluorescent compound in accordance with exemplary embodiments of the present invention;

(3) FIG. 2 illustrates a melting curve shown when DNA is synthesized using the fluorescent compound in accordance with exemplary embodiments of the present invention;

(4) FIG. 3 illustrates a C.sub.q value when DNA is synthesized using a reagent using the fluorescent compound in accordance with exemplary embodiments of the present invention;

(5) FIG. 4 illustrates comparison of fluorescence intensities shown when DNA is synthesized using a fluorescent compound of embodiments of the present invention and a reference material;

(6) FIG. 5 illustrates a difference in melting curve shown when DNA is synthesized using a fluorescent compound of embodiments of the present invention and a reference material;

(7) FIG. 6 illustrates C.sub.q values when DNA is synthesized using a fluorescent compound of embodiments of the present invention and a reference material;

(8) FIG. 7 illustrates changes in fluorescence intensity according to concentrations of a compound of embodiments of the present invention and a reference material before reaction when DNA is synthesized using the compound of embodiments of the present invention and the reference material;

(9) FIG. 8 illustrates changes in melting curve according to concentrations of the compound of embodiments of the present invention and the reference material before reaction when DNA is synthesized using the compound of embodiments of the present invention and the reference material;

(10) FIG. 9 illustrates C.sub.q values according to concentrations of a compound of embodiments of the present invention and a reference material before reaction when DNA is synthesized using the compound of embodiments of the present invention and the reference material;

(11) FIG. 10 illustrates electrophoresis results before reaction when DNA is synthesized using the compound of embodiments of the present invention and the reference material;

(12) FIG. 11 illustrates changes in fluorescence intensity according to concentrations of a compound of embodiments of the present invention and a reference material after reaction when DNA is synthesized using the compound of embodiments of the present invention and the reference material;

(13) FIG. 12 illustrates changes in melting curve according to concentrations of a compound of embodiments of the present invention and a reference material after reaction when DNA is synthesized using the compound of embodiments of the present invention and the reference material;

(14) FIG. 13 illustrates C.sub.q values according to concentrations of a compound of embodiments of the present invention and a reference material after reaction when DNA is synthesized using the compound of embodiments of the present invention and the reference material; and

(15) FIG. 14 illustrates electrophoresis results after reaction when DNA is synthesized using the compound of embodiments of the present invention and the reference material.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(16) Hereinafter, a preparation method of a fluorescent compound in accordance with exemplary embodiments of the present invention and the fluorescence efficiency and the like of the compound will be described in detail using Examples of the present invention.

(17) Exemplary embodiments of the present invention provide a compound represented by the following Chemical Formula 1.

(18) ##STR00002##

(19) Wherein,

(20) X is oxygen or sulfur, and

(21) R is —SO.sub.2CH(CH.sub.2), —CH.sub.2N(CH.sub.3).sub.2, or unsubstituted alkyl having 5 to 10 carbon atoms.

(22) Specific compounds included in exemplary embodiments of the present invention may be compounds of Compound 8, Compound 9, Compound 10, Compound 15, Compound 16, and Compound 17 below.

(23) ##STR00003## ##STR00004##

(24) Hereinafter, exemplary embodiments of the present invention will be described in more detail through Examples. However, the following Examples are not to limit the scope of the present invention and will be described to help in the understanding of the present invention.

(25) A preparation method of a compound in accordance with exemplary embodiments of the present invention is as follows.

(26) ##STR00005##

(27) 2-(methylthio)benzoxazole (5 ml, 37.5 mmol) and methyl p-toluensulfonate (11.3 ml, 75.1 mmol) were dissolved in 5 ml of dimethylformamide, and then stirred at 150° C. for 1 hour under a nitrogen atmosphere. After the reaction was completed, particles were precipitated with ethylacetate, separated using a centrifuge, and then dried in a vacuum dryer to obtain Compound 1 above.

(28) LC/MS, calculated value of C.sub.9H.sub.10NOS.sup.+180.2, measured value of 180.1

(29) ##STR00006##

(30) 1-methyl-2-pyrroldinone (50 ml, 0.520 mol) was dissolved in 80 ml of hydrochloride, and then stirred at 130° C. for 12 hours under a nitrogen atmosphere. After the reaction was completed, particles were precipitated using 500 ml of acetone after drying under reduced pressure. The precipitated particles were filtered under reduced pressure and then dried to obtain Compound 2 above.

(31) LC/MS, calculated value of C.sub.5H.sub.11NO.sub.2117.1, measured value of 118.1

(32) ##STR00007##

(33) 4-methyl carbostyril (10 g, 32.9 mmol), copper powder (24 g, 37.7 mmol), and potassium carbonate (8.7 g, 62.9 mmol) were dissolved in 80 ml of Iodobenzene and then stirred at 200° C. for 48 hours under a nitrogen atmosphere. After the reaction was completed, the mixture was extracted 3 times with ethylacetate and distilled water. An organic layer was collected, concentrated after removing moisture with magnesium sulfate, and then purified by silica gel chromatography (eluent: 50 to 100% EA-Hexane) to obtain Compound 3 above.

(34) LC/MS, calculated value of C.sub.16H.sub.13NO235.2, measured value of 236.3

(35) ##STR00008##

(36) Phosphorus(V) oxychloride (187 μl, 2 mmol) was dissolved in 100 μl of dimethylformamide in an ice bath, Compound 3 was dissolved in 3.5 ml of dichloromethane, added to the reaction solution, and then stirred at 60° C. for 24 hours under a nitrogen atmosphere to obtain Compound 4 above.

(37) LC/MS, calculated value of C.sub.16H.sub.14ClN255.7, measured value of 254.1

(38) ##STR00009##

(39) Compound 1 (360 mg, 2 mmol) and triethylamine (1.3 ml, 10 mmol) were added to the reaction solution of Compound 4 and then stirred at 60° C. for 2 hours. After cooling to room temperature, particles were precipitated with diethyl ether, and then separated using a centrifuge. The separated particles were purified by silica gel chromatography (eluent: DCM:MeOH:EA=4:1:3) to obtain Compound 5 above.

(40) LC/MS, calculated value of C.sub.24H.sub.18ClN.sub.2O.sup.+385.8, measured value of 385.1

(41) ##STR00010##

(42) Compound 2 (292 mg, 2.49 mmol) and t-butylmethylsilyl chloride (311 mg, 2.07 mmol) were dissolved in 20 ml of 1,2-dichloroethane, and then added with triethylamine (1.34 ml, 9.62 mmol) and stirred at room temperature for 4 hours. Compound 5 (275 mg, 0.711 mmol) was added to the reaction solution, and then stirred at 60° C. for 1 hour under a nitrogen atmosphere. After cooling to room temperature, particles were precipitated with diethyl ether, separated using a centrifuge, and then dried to obtain Compound 6 above.

(43) LC/MS, calculated value of C.sub.29H.sub.28N.sub.3O.sub.3.sup.+466.5, measured value of 466.2

(44) ##STR00011##

(45) Compound 6 (70 mg, 0.15 mmol) and N,N,N′,N′-tetramethyl-O—(N-succinimidyl)uronium tetrafluoroborate (45.2 mg, 0.15 mmol) were dissolved in 4 ml of dimethylformamide, and then added with triethylamine (104.5 μl, 0.75 mmol) and stirred at room temperature for 30 minutes. Particles were precipitated with diethyl ether, separated using a centrifuge, and then dried to obtain Compound 7 above.

(46) LC/MS, measured value of C.sub.33H.sub.31N.sub.4O.sub.5.sup.+563.6, measured value of 563.3

(47) ##STR00012##

(48) Compound 7 (178 mg, 0.317 mmol) and 2-(2-chloroethylsulfonyl)ethanamine hydrochloride (132 mg, 0.634 mmol) were dissolved in 5 ml of dimethylformamide, and then added with N,N-diisopropylethylamine (552 μl, 3.17 mmol) and stirred for 24 hours at room temperature. Particles were precipitated with diethyl ether, separated using a centrifuge, and then dried to obtain Compound 8 above.

(49) LC/MS, calculated value of C.sub.33H.sub.35N.sub.4O.sub.4S.sup.+583.7, measured value of 583.3

(50) ##STR00013##

(51) Compound 7 (207 mg, 0.367 mmol) and 3-(dimethylamino)-1-propylamine (139 μl, 1.10 mmol) were dissolved in 5 ml of dimethylformamide, and then added with N,N-diisopropylethylamine (639 μl, 3.67 mmol) and stirred for 24 hours at room temperature. Particles were precipitated with diethyl ether, separated using a centrifuge, and then dried to obtain Compound 9 above.

(52) LC/MS, calculated value of C.sub.34H.sub.40N.sub.5O.sub.2.sup.+550.7, measured value of 550.4

(53) ##STR00014##

(54) Compound 7 (247 mg, 0.438 mmol) and octylamine (217 μl, 1.31 mmol) were dissolved in 5 ml of dimethylformamide, and then added with N,N-diisopropylethylamine (762 μl, 4.38 mmol) and stirred for 24 hours at room temperature. Particles were precipitated with diethyl ether, separated using a centrifuge, and then dried to obtain Compound 10 above.

(55) LC/MS, calculated value of C.sub.37H.sub.45N.sub.4O.sub.2.sup.+577.7, measured value of 577.4

(56) ##STR00015##

(57) 2-(methylthio)benzoxazole (5 g, 0.028 mol) and iodomethane (6.9 ml, 0.110 mol) were dissolved in 20 ml of methanol, and then stirred at 50° C. for 24 hour under a nitrogen atmosphere. After the reaction was completed, the reaction solution was dried under reduced pressure, and then particles were precipitated with diethyl ether. The precipitated particles were filtered under reduced pressure and then dried in vacuum to obtain Compound 11 above.

(58) LC/MS, calculated value of C.sub.9H.sub.10NS.sub.2.sup.+196.3, measured value of 196.2

(59) ##STR00016##

(60) Compound 11 (392 mg, 2 mmol) and triethylamine (0.28 ml, 2 mmol) were added to the reaction solution of Compound and then stirred at room temperature for 12 hours. Particles were precipitated with diethyl ether, and then separated using a centrifuge. The separated particles were purified by silica gel chromatography (eluent: CHCl.sub.3:MeOH:EA=3:1:3) to obtain Compound 12 above.

(61) LC/MS, calculated value of C.sub.24H.sub.18ClN.sub.2S.sup.+401.9, measured value of 401.1

(62) ##STR00017##

(63) Except for using Compound 12 instead of Compound 5, Compound 13 above was obtained through the same process as the method of synthesizing Compound 6.

(64) LC/MS, calculated value of C.sub.29H.sub.28N.sub.3O.sub.2S.sup.+482.6, measured value of 482.2

(65) ##STR00018##

(66) Compound 14

(67) Except for using Compound 13 instead of Compound 6, Compound 14 above was obtained through the same process as the method of synthesizing Compound 7.

(68) LC/MS, calculated value of C.sub.33H.sub.31N.sub.4O.sub.4S.sup.+579.6, measured value of 579.2

(69) ##STR00019##

(70) Except for using Compound 14 instead of Compound 7, Compound 15 above was obtained through the same process as the method of synthesizing Compound 8.

(71) LC/MS, calculated value of C.sub.33H.sub.35N.sub.4O.sub.3S.sub.2.sup.+599.7, measured value of 599.3

(72) ##STR00020##

(73) Except for using Compound 14 instead of Compound 7, Compound 16 above was obtained through the same process as the method of synthesizing Compound 9.

(74) LC/MS, calculated value of C.sub.34H.sub.40N.sub.5OS.sup.+566.7, measured value of 566.9

(75) ##STR00021##

(76) Except for using Compound 14 instead of Compound 7, Compound 17 above was obtained through the same process as the method of synthesizing Compound 10.

(77) LC/MS, calculated value of C.sub.37H.sub.45N.sub.4OS.sup.+593.8, measured value of 594.0

(78) Examples: Preparation of Contrast Agent Composition Using Compound of the Present Invention

(79) (1) Preparation of Reagent for Real-Time PCR Analysis

(80) Compounds 8, 9, 10, 15, 16, and 17 were dissolved in dimethyl sulfoxide to prepare 5 mM of a stock solution. The prepared stock solution was diluted in a Tris-EDTA buffer (pH 7.5) and prepared at a concentration of 200 uM.

(81) For Reference material 1, 10,000× of the stock solution was diluted to a concentration of 20× through a Tris-EDTA buffer (pH 7.5). For Reference material 2, 20,000× of the stock solution was diluted to a concentration of 20× through a Tris-EDTA buffer (pH 7.5).

(82) (2) Compound Analysis Through Real-Time RCR

(83) Reference materials 1 (Evagreen) and 2 (Invitrogen Sybr Green) and Compounds 8, 9, 10, 15, 16, and 17 were compared and verified by an intercalating method using a double-stranded DNA binding dyes through real-time PCR. cDNA extracted from HeLa cells was used, and b-actin (Forward 5′-CAT CGA GCA CGG CAT CGT CA-3′, Reverse 5′-TAG CAC AGC CTG GAT AGC AAC-3′) was used as a primer. Real-time PCR reaction was performed using a CFX96 touch real-time PCR detection system (BIO-RAD). The PCR mixture consisted of a total 20 uL of reaction solution containing 1 uL of a forward primer, 1 uL of a reverse primer, 10 uL of a PCR master mix (TAKARA), 1 uL of a reference material and each compound (Compounds 8, 9, 10, 15, 16, 17), and 1 uL of template cDNA. PCR conditions were as follows: (a) In a pre-denaturing step, 1 minute at 95° C. (b) 45 cycles are configured as a cycle consisting of at 95° C. for 15 seconds, 58° C. for 30 seconds and 72° C. for 30 seconds, and a fluorescence wavelength of 497 nm was detected in a cycle of 30 seconds at 72° C.

(84) FIG. 1 illustrates that fluorescence signals were not strongly observed during DNA synthesis using Compounds 8, 9, and 10, and the C.sub.q values were not much confirmed as in FIG. 3. On the other hand, it can be seen that Compounds 15, 16, and 17 emit visible fluorescence by binding to DNA during DNA amplification as illustrated in FIG. 1. Compounds 15 and 17 were not much strong in fluorescence, and the C.sub.q value of Compound 17 was also hardly confirmed. On the other hand, it can be seen that Compound 16 shows a very strong fluorescence intensity and the C.sub.q value is also the fastest.

(85) FIG. 2 shows a melting curve and shows the same result as the above result.

(86) As illustrated in FIG. 4, Compound 16 showed a fluorescence intensity that was two times higher than that of Reference materials 1 and 2, and a C.sub.q value that was faster than that of Reference material 1 was confirmed.

(87) FIG. 5 shows a melting curve and shows the same result as the above result.

(88) (3) Stability Analysis of Compound

(89) Stability analysis was performed by storing Reference materials 1 (Evagreen) and 2 (Invitrogen Sybr Green) and Compound 16 in an incubator at 60° C. The three materials were stored in an incubator at 60° C. for 3 days, and the fluorescence signals for each template concentration were analyzed by real-time PCR analysis as in (1) above, and the PCR product was electrophoresed to analyze whether normal DNA synthesis was achieved. After real-time-PCR, synthetic DNA and a 6× Loading dye (BioActs) were mixed, and then electrophoresis was performed for 35 minutes on a 2% agarose gel (Simga-aldrich).

(90) FIGS. 7, 8, 9, and 10 illustrate real-time PCR and electrophoresis results before reaction at 60° C.

(91) FIGS. 11, 12, 13, and 14 showed no significant change in Reference material 1 and Compound 16 as a result of reaction at 60° C. for 3 days. On the other hand, it was confirmed that the fluorescence intensity of Reference material 2 decreased by about half, and Compound 16 was a more stable material than Reference material 2. In addition, as a result of electrophoresis, Compound 16 showed clearer DNA synthesis results than Reference materials 1 and 2.

(92) From the results, it can be seen that the compounds provided by the present invention have effects on the DNA synthetic fluorescent dye less than the conventional fluorescent compounds, and thus, the compounds have excellent stability.

(93) The present invention is not limited by the above-described embodiments, and various modifications and changes can be made by those skilled in the art and may be used in various biological and chemical fields, and are included in the spirit and scope of the present invention as defined in the appended claims.