WO2020067854A1 - Novel compounds for synthesizing photoreactive nucleic acid - Google Patents
Novel compounds for synthesizing photoreactive nucleic acid Download PDFInfo
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- WO2020067854A1 WO2020067854A1 PCT/KR2019/012889 KR2019012889W WO2020067854A1 WO 2020067854 A1 WO2020067854 A1 WO 2020067854A1 KR 2019012889 W KR2019012889 W KR 2019012889W WO 2020067854 A1 WO2020067854 A1 WO 2020067854A1
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 75
- 150000007523 nucleic acids Chemical class 0.000 title claims description 111
- 108020004707 nucleic acids Proteins 0.000 title claims description 108
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/655—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms
- C07F9/6552—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a six-membered ring
- C07F9/65522—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a six-membered ring condensed with carbocyclic rings or carbocyclic ring systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/655—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Definitions
- the present invention relates to a method for producing a novel compound for photoreactive nucleic acid synthesis and its use, and more specifically, to a group comprising a group capable of polymer synthesis and conjugation with other molecules on the coumarin based on coumarin having excellent photoreactivity. It relates to a compound for synthesizing a photoreactive nucleic acid.
- SNP single nucleotide polymorphism
- a single nucleotide of a gene (and other types of gene polymorphisms such as small insertions / deletions) provides a variety of information was also thought to be attributed to the increased interest.
- a single nucleotide is also involved in, or at least represents, an important trait in human, plant and animal species.
- the photoreactive nucleic acid is a chemical bond that is broken by light and is introduced into the nucleic acid, and can be used for nucleic acid sequence analysis, nucleic acid position analysis, and the like in a single cell.
- the ortho-nitrobenzyl compound used in the conventional photoreactive nucleic acid is sensitive to only light of the UV wavelength, and has a shallow depth of penetration and damage to the cell. There is a problem that is difficult to observe in the region.
- the present inventors used a coumarin derivative that is sensitive to light in the near-infrared region as a result of diligent efforts to solve the conventional problems, and introducing a functional group capable of polymer synthesis and conjugation with other molecules to the photoreactive functional group without additional steps. It has been confirmed that a nucleic acid capable of synthesis and conjugation with other molecules can be provided.
- the present invention was completed by confirming that the conventional problems were improved, including a novel coumarin compound capable of being used for nucleic acid synthesis and capable of polymer synthesis and conjugation with other molecules.
- One object of the present invention is to provide a compound represented by the following formula (1).
- R 1 is hydrogen or C 1-6 alkyl
- R 2a to R 2b are each independently C 1-6 alkyl
- n 1 is an integer selected from 0 to 40
- n 2 is an integer selected from 0 to 40
- the m is an integer selected from 1 to 3.
- Another object of the present invention is to provide a composition for synthesizing a photoreactive nucleic acid comprising a compound of Formula 1.
- Another object of the present invention is a first step of preparing a first complex in which a first nucleic acid is bound to a phosphoramidite moiety of Formula 1 by reacting a compound represented by the following Formula 1 with a first nucleic acid: It provides a method for producing a nucleic acid, comprising a second step of preparing a second complex to form a double-stranded nucleic acid of complementary sequence by contacting the complex with the second nucleic acid.
- the compound of the present invention solves the problem that the ortho-nitrobenzyl compound, which has been used in the conventional photoreactive nucleic acid, has a shallow penetration depth and can damage cells by responding only to UV wavelength light, and the compound of the present invention analyzes the nucleic acid position When used in the nature of the UV wavelength, it solves the problem that it was difficult to observe in a narrow area due to a large focal volume.
- the present invention uses a compound containing a coumarin derivative that is sensitive to light in the near-infrared region, thereby providing an effect of deep penetration and less cell damage.
- a separate process after synthesis of the photoreactive nucleic acid, which was necessary to make the photoreactive nucleic acid into a form capable of polymer polymerization or conjugation with other molecules, is not required, thereby providing an economical effect.
- Figure 2 is a graph showing the MS analysis of the complex of the compound of formula 2 of the present invention.
- a of FIG. 4 is a structural formula showing the function of each functional group of Formula 1
- B is a schematic diagram showing the process of photolysis of the nucleic acid linked to the compound of the present invention by light in the near-infrared region
- C is immediately after photolysis and fluorescence quenching
- the confocal microscopy image of and D is a confocal microscopy image after staining a hydrogel containing nucleic acid with a new fluorescent probe after photolysis and fluorescence quenching.
- One aspect of the present invention for achieving the above object provides a compound represented by the formula (1).
- R 1 is hydrogen or C 1-6 alkyl
- R 2a to R 2b are each independently C 1-6 alkyl
- n 1 is an integer selected from 0 to 40
- n 2 is an integer selected from 0 to 40
- the m of Y is an integer selected from 1 to 3.
- Another aspect of the present invention for achieving the above object is to provide a composition for synthesizing a photoreactive nucleic acid comprising a compound of Formula 1 below.
- R 1 is hydrogen or C 1-6 alkyl
- R 2a to R 2b are each independently C 1-6 alkyl
- n 1 is an integer selected from 0 to 40
- n 2 is an integer selected from 0 to 40
- the m is an integer selected from 1 to 3.
- Another aspect of the present invention for achieving the above object is to react the compound represented by the following formula (1) and the first nucleic acid to form a first complex in which the first nucleic acid is bound to the phosphoramidite moiety of the formula (1).
- a first step of manufacturing and a second step of making a second complex of contacting the first complex with a second nucleic acid to form a nucleic acid double strand of complementary sequence are provided.
- R 1 is hydrogen or C 1-6 alkyl
- R 2a to R 2b are each independently C 1-6 alkyl
- n 1 is an integer selected from 0 to 40
- n 2 is an integer selected from 0 to 40
- the m is an integer selected from 1 to 3.
- the compound may be used for nucleic acid synthesis, nucleic acid separation and complex formation, and the ortho-nitrobenzyl compound that has been used in the existing photoreactive nucleic acid is sensitive to UV light only and has a shallow penetration depth and damages cells.
- the compound of the present invention is used for nucleic acid position analysis, it solves a problem that it is difficult to observe in a narrow region due to the large focus volume due to the nature of the UV wavelength, and is sensitive to light in the near infrared region as well as UV.
- nucleic acid synthesis Solid-phase oligonucleotide synthesis
- nucleotide synthesis Solid-phase oligonucleotide synthesis
- phosphate ester of the nucleoside sugar moiety is synthesized by complementary sequence interaction, respectively.
- complex formation may mean that the first nucleic acid and the second nucleic acid of the present invention form a double-stranded nucleic acid by complementary sequence action, wherein the second nucleic acid is a part of the first nucleic acid or It may be a single-stranded nucleic acid having a sequence complementary to all or a mixture of A, G, C and T nucleic acid monomers, wherein the second nucleic acid is between the nucleic acid including a sequence complementary to the first nucleic acid or a sequence similar thereto. According to the interaction, it may mean the second complex.
- nucleic acid separation means that a nucleic acid and another compound are separated from a complex in which a nucleic acid and other compounds are bound.
- the third complex of the present invention when the third complex of the present invention is irradiated with light, it can be said that only the nucleic acid can be obtained by being separated from the compound and composition represented by Chemical Formula 1.
- the compound may be represented by the following Chemical Formula 2, Chemical Formula 3, or Chemical Formula 4.
- R 1 is hydrogen or C 1-6 alkyl
- R 2a to R 2b are each independently C 1-6 alkyl
- n is an integer selected from 0 to 40
- n is an integer selected from 1 to 3.
- coumarin of the present invention is an organic compound belonging to the heterocyclic family, and is a colorless crystal in appearance. It is known as an aromatic component of tonka bean and is used as a fragrance. Also called benzo- ⁇ pyrone, o-oxycinnamic acid Corresponds to lactone. Formula C 9 H 6 O 2 . It is a very small amount of aroma component of tonka bean, but it is contained in various plants, especially fruits. It is a colorless crystal and has a molecular weight of 146.15, a melting point of 71 ° C, a boiling point of 301.7 ° C, and a specific gravity of 0.935. It is difficult to dissolve in cold water, but dissolves in hot water. It means that salicylaldehyde and acetic anhydride are obtained by condensation in the presence of anhydrous potassium acetate, which is used as a fragrance and is often used as a fragrance for a substituent.
- the coumarin may be one containing 7-hydroxy-4-methylcoumarin.
- the compound of the present invention may be a compound containing 7-hydroxy-4-methylcoumarin in the parent nucleus.
- the compounds and compositions of the present invention include coumarin, and coumarin is excellent in photoreactivity, so it can be activated by absorbing it in a near-infrared light with a high efficiency, i.
- the vinyl group included in the compounds and compositions of the present invention can be combined with the polymer contained in the polymer network gel, and the third complex fixed to the polymer can be easily fixed to irradiate light.
- the coumarin may be photodegradable.
- photolysis of the present invention is a separation that occurs by absorbing light in response to light, and is used for direct photolysis that is absorbed by light-absorbing substances and energy transfer or chemical reaction from excitation light-sensitizers generated by absorbing light.
- photosensitive separation separated by a reactant, and it means that one molecule absorbs light and is separated into two or more components.
- the compound of the present invention includes a coumarin linker as a photoreactive material, and coumarin converts light energy into chemical energy, and can be photodegraded to be converted into a material having a lower molecular weight by photoreaction by photochemical action.
- the compound of the present invention was photoreacted to separate the nucleic acid from the third complex to obtain only the nucleic acid.
- composition may be to combine with a polymer network gel through a vinyl group included in the compound of formula (1).
- the compound of the present invention may be copolymerized when polymerized with a polymer network gel including a vinyl group.
- the polymer network gel to which the compound of the present invention is immobilized may be a medium capable of synthesizing and isolating nucleic acids.
- the compound of the present invention may be fixed to a polymer network including a vinyl group or conjugated with a material capable of forming a chemical bond by reaction with a vinyl group.
- the polymer network gel may be a material capable of reacting with a vinyl group.
- composition may be synthesized with a nucleic acid through a phosphoramidite moiety included in the compound of Formula 1.
- phosphoramidite moiety (phosphoramidite moiety) of the present invention may indicate a portion represented by the following formula 1-1 except for the vinyl group in the formula 1 of the present invention, through this portion to bind to the nucleic acid Can form:
- the compound of Formula 1 may include a vinyl group at both ends and an embedded phosphoramidite moiety, and may be combined with a polymer network gel in the vinyl group, and binds to the first nucleic acid in the phosphoramidite moiety portion.
- the first nucleic acid and the second nucleic acid can form a double-stranded nucleic acid having a complementary sequence to form a second complex, and can form a complex in the form of a polymer network gel-compound-nucleic acid represented by the third complex of the present invention. have.
- the composition may be to separate nucleic acids synthesized through diphoton absorption by coumarin contained in the compound of Formula 1 when irradiated with infrared rays.
- composition of the present invention may be photoreactive to infrared light including coumarin, and the nucleic acid combined with the composition of the present invention may be separated by reacting to light having an infrared wavelength.
- the second nucleic acid may be a single-stranded nucleic acid having a sequence complementary to some or all of the first nucleic acid or a mixture of A, G, C and T nucleic acid monomers.
- the third step of forming a third complex by bonding the polymer network gel to the vinyl group of Formula 1 before or after the second step may be further included.
- the third step may further include the step of isolating the nucleic acid by irradiating the compound with light.
- the first complex may be represented by Formula 1-first nucleic acid
- the second complex may be formed in the form of Formula 1-second nucleic acid-second nucleic acid
- the third complex is a polymer network gel-formula It can be formed in the form of 1-first nucleic acid-second nucleic acid.
- the coumarin contained in Chemical Formula 1 is photoreacted by irradiating light to be separated into a polymer network gel-form of Formula 1 and a first nucleic acid-second form of nucleic acid. To obtain a nucleic acid.
- the light may be ultraviolet, visible or infrared.
- UV ultraviolet rays
- UV ultraviolet light having a wavelength ranging from 10 to 400 nm (energy range 3 eV to 124 eV), which is shorter than visible light and longer than X-rays, and is almost transparent to glass. It cannot, and the air does not pass well. It is a high-energy wave with a high frequency, which means that the chemical action is strong enough to partially destroy or separate molecules in life.
- visible light in the present invention is light having a wavelength range perceived by the eye. Physical light is a spectrum within the wavelength limit of the range perceived as color to the eye, and refers to electromagnetic waves having a wavelength in the range of approximately 380 to 780 nm (nanometer).
- infrared is an electromagnetic wave having a longer wavelength than visible light and falling in a range of 0.75 ⁇ m to 1 mm. Dispersion of light emitted from sunlight or incandescent objects into the spectrum is called infrared because it is located farther than the end of the red spectrum. Infrared rays with a wavelength of 0.75 to 3 ⁇ m are called near infrared, those with 3-25 ⁇ m are simply called infrared, and those with a wavelength of 25 ⁇ m or more are called far infrared. It is characterized by having a stronger thermal action than visible light or ultraviolet light, and for this reason, it is also referred to as heat rays.
- the compounds of the present invention respond to UV (UV) only, including coumarins that respond not only in the UV but also in the near-infrared region, resulting in shallow penetration depth and damage to the cells. You can solve this difficult problem.
- the compound of the present invention reacts in the UV as well as near-infrared rays, it reacts only to UV, causing a problem that shallow penetration depth and damage to cells and difficulty in observing in a small volume are difficult.
- an economic effect was confirmed because a separate process after photoreactive nucleic acid synthesis, which was necessary to make the photoreactive nucleic acid polymerizable or conjugated with other molecules, is not required.
- Step a First, compound 2 was prepared (compound 2 represented by 2 in scheme 1). 7-hydroxy-4-methylcoumarin (0.30 g, 1.70 mmol) was dissolved in acetone (18 mL) and K 2 CO 3 (0.259 g, 1.87 mmol) and crown ether (0.450 g, 1.70 mmol) were added. After stirring for 1 hour in a reflux apparatus, 2-tertbutyloxycarbonyl aminoethyl bromide (0.42 g, 1.87 mmol) was added. After stirring for 18 hours in a reflux device, it was filtered and concentrated. Purification by silica gel flash column chromatography gave compound 2 as a white solid. (0.483 g, 88% yield)
- Step b Next, compound 3 was prepared (b reaction to produce compound 3 represented by 3 in scheme 1).
- Step c Compound 4 was prepared (c-reaction to prepare compound 4 represented by 4 in Scheme 1).
- the crude compound 3 was dissolved in water (4 mL) and THF (4 mL) and NaIO 4 (0.60 g, 2.8 mmol) was added. After stirring at room temperature for 2 hours, filtered and concentrated under reduced pressure. Then saturated NaHCO 3 solution was added and extracted with CH 2 Cl 2 . The obtained organic layer was dried over anhydrous MgSO 4 , filtered and concentrated under reduced pressure to obtain crude compound 4. It was used for the next reaction without a separate purification process.
- Step d Compound 5 was prepared (d reaction to produce compound 5 represented by 5 in Scheme 1).
- Step e Compound 6 was prepared (e-reaction to prepare compound 6 represented by 6 in Scheme 1).
- Step f Preparation of compound 7 (f reaction to prepare compound 7 represented by 7 in scheme 1)
- Step g Formula 2 was prepared (g reaction to prepare Formula 2 represented by 8 in Scheme 1).
- the compound 7 (0.2 g, 0.66 mmol) was dissolved in anhydrous acetonitrile (3.5 mL), followed by 1H-triazole (1.5 mL, 0.72 mmol) and 2-cyanoethyl N, N, N ', N'-tetra Isopropylphosphodiamidite (0.42 mL, 1.3 mmol) was added slowly. After stirring at room temperature for 16 hours, filtered and concentrated under reduced pressure. Then ethyl acetate was added and washed with saturated NaHCO 3 solution. The saturated NaHCO 3 solution was extracted again with ethyl acetate.
- Example 2 nucleic acid synthesis and separation method
- a first complex in which the first nucleic acid is bound to the phosphoramidite moiety of Formula 1 was prepared by reacting the first nucleic acid with Formula 2 prepared in Example 1, and the second compound contained in the biological sample in the first complex.
- Nucleic acid was synthesized by contacting the nucleic acid to form a nucleic acid double strand having a complementary sequence, and a polymer network gel was bonded to a vinyl group of Formula 2 to form a third complex.
- the third complex is irradiated with a near-infrared 780nm wavelength biphoton laser at 25% output, and nucleic acid is separated from the coumarin of Formula 2 through photoreaction to obtain nucleic acid.
- n and m in Formula 2 are integers 1, and R 1 is methyl R 2a and R 2b is isopropyl.
- a functional group that is bonded to a polymer including a vinyl group was identified, a functional group capable of photoreaction including coumarin, and a portion bound to a nucleic acid, including a phosphoramidite moiety. I could confirm.
- Example 2 The composite prepared in Example 2 was analyzed for mass using MALDI-TOF before and after ultraviolet irradiation.
- the poly (dT) 20 nucleic acid linked to Formula 2 is copolymerized with an acrylamide monomer and an APS initiator to form a gel composed of a polymer network, and then the poly (dT) present in the gel is used using a fluorescent poly (dA) probe. Dyed.
- a specific part of the gel ( ⁇ in FIG. 4C) was irradiated with a near-infrared 780 nm wavelength photon laser to induce photolysis of Formula 2, and the other part of the gel (# in FIG. 4C) was used with a single photon laser of 488 nm wavelength. Fluorescence of the poly (dA) probe was quenched. As shown in the schematic diagram of FIG. 4B, the fluorescence reaction was observed when the existing fluorescent probe was removed through heating and a new fluorescent probe was added.
- a of FIG. 4 showing the composite of the present invention was subjected to a photolysis experiment by irradiating two wavelengths as shown in the schematic diagram of B of FIG. 4.
- the # portion can be quenched by quenching only fluorescence with a single photon laser of 488 nm wavelength, and the ⁇ portion was irradiated with a 780 nm wavelength diphoton laser to confirm that only the nucleic acid in the complex can be separated and photodegraded. . Therefore, C in FIG.
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Abstract
The present invention relates to a compound represented by chemical formula 1, and nucleic acid synthesis using same with less cell damage due to sensitivity even in a near-infrared light region, in which an additional step after synthesis and purification is not required, thus providing an economical effect. [Chemical formula 1] In chemical formula 1, R1 is hydrogen or a C1-6 alkyl, R2a and R2b are each independently a C1-6 alkyl, n1 is an integer selected from 0 to 40, n2 is an integer selected from 0 to 40, L is [Chemical formula 2], and m is an integer selected from 1 to 3.
Description
본 발명은 광반응성 핵산 합성을 위한 신규 화합물 이의 제조방법 및 이의 용도에 관한 것으로, 더욱 자세하게는 광반응성이 우수한 쿠마린을 기반으로 상기 쿠마린 상에 고분자 합성 및 다른 분자와의 접합이 가능한 그룹을 포함하는 광반응성 핵산 합성용 화합물에 관한 것이다.The present invention relates to a method for producing a novel compound for photoreactive nucleic acid synthesis and its use, and more specifically, to a group comprising a group capable of polymer synthesis and conjugation with other molecules on the coumarin based on coumarin having excellent photoreactivity. It relates to a compound for synthesizing a photoreactive nucleic acid.
특정 핵산 서열의 검출의 관심이 급속히 증대하고 있다. 이 관심은 최근 개시된 인간게놈의 뉴클레오티드 서열안 및 그 중 및 많은 다른 생물, 다량의 단일 뉴클레오티드 다형(SNP) 게놈의 존재뿐만 아니라 AFLP 등 마커 기술 및 예를 들면 유전학적 유전성 질환의 지표로서의 특정 핵산 서열의 검출 타당성 일반적 인식에서도 발생하고 있다.The interest in detecting specific nucleic acid sequences is rapidly increasing. This interest is not only present in the nucleotide sequence of recently disclosed human genomes and among them and many other organisms, the presence of large amounts of single nucleotide polymorphism (SNP) genomes, but also marker techniques such as AFLP and specific nucleic acid sequences as indicators of genetic genetic diseases, for example The detection feasibility of detection also occurs in general recognition.
유전자의 단일 뉴클레오티드(및 작은 삽입/결실 등 다른 종류의 유전자 다형)가 다양한 정보를 제공한다고 하는 인식은 이 관심의 증대로 인하다고도 생각되었다. 현재, 이들의 단일 뉴클레오티드 치환이 예를 들면 사람의 다수의 단인자 및 다인자 유전성 질환의 주된 원인의 하나이며 또는 식물 및 가축종의 성능 형질 등 복잡한 표현형 발현에 관여하고 있는 것이 일반적으로 인식되어 있다. 따라서, 단일 뉴클레오티드는 많은 경우에 사람, 식물 및 동물종에 있어서의 중요한 형질에도 관련되어, 또는 적어도 이것을 나타낸다.The perception that a single nucleotide of a gene (and other types of gene polymorphisms such as small insertions / deletions) provides a variety of information was also thought to be attributed to the increased interest. Currently, it is generally recognized that their single nucleotide substitution is one of the main causes of multiple monofactorial and multifactorial genetic diseases in humans, or is involved in the expression of complex phenotypes such as performance traits of plant and livestock species. . Thus, a single nucleotide is also involved in, or at least represents, an important trait in human, plant and animal species.
이들의 단일 뉴클레오티드 치환 및 분석은 결과적으로 가능한 한 넓은 점에서 의학 및 농업으로 광범위하게 미치는 의의를 가지는 대량의 정보를 가져온다. 예를 들면 이들의 발전이 결과적으로 환자 특이적 의약품을 가져오는 것이 일반적으로 상정되어 있다. 이들의 유전자 다형을 분석하기 위해, 얻어진 데이터의 품질을 크게 훼손하지 않고 다수의 샘플 및 다수의(주로) SNP 높은 처리량 방법에서의 처리를 가능하게 하는 충분히 확실하고 또한 신속한 방법의 필요성이 증대되어 있다. 기존의 배열 핵산 분석에 사용되는 주된 방법의 하나는 2개의 프로브를 표적 배열과 어닐링하고 프로브가 표적 배열과 인접해 혼성화되면, 프로브를 라이게이트하는 스텝에 기초한다. 이 컨셉은 올리고뉴클레오티드 결찰(Ligation) 어세이 또는 올리고뉴클레오티드 결찰(Ligation) 증폭(OLA)으로서 일반적으로 나타나고 있다.Their single nucleotide substitution and analysis results in a large amount of information that has a broad implication for medicine and agriculture as broadly as possible. It is generally assumed, for example, that their development will result in patient-specific medicines. To analyze their genetic polymorphisms, there is an increasing need for a method that is sufficiently reliable and rapid that enables processing in multiple samples and multiple (mainly) SNP high throughput methods without significantly degrading the quality of the data obtained. . One of the main methods used in conventional array nucleic acid analysis is based on the step of annealing two probes with the target array and ligating the probes when the probes hybridize adjacent to the target array. This concept is commonly seen as an oligonucleotide ligation assay or oligonucleotide ligation amplification (OLA).
이러한 핵산 분석을 위해 광반응성 화합물을 사용하는 방법이 제안되고 있다. 광반응성 핵산은 빛에 의해 끊어지는 화학 결합을 핵산에 도입한 것으로 단일 세포에서의 핵산 서열 분석, 핵산 위치 분석 등에 이용될 수 있다. 그러나 기존의 광반응성 핵산에 사용된 오르토-니트로벤질 화합물은 UV 파장의 빛에만 감응하여 침투 깊이가 얕고 세포에 손상을 줄 수 있고, 핵산 위치 분석에 사용될 경우 UV 파장의 특성 상 초점 부피가 커 좁은 영역에서의 관찰이 어려운 문제점이 있다.A method of using a photoreactive compound for such nucleic acid analysis has been proposed. The photoreactive nucleic acid is a chemical bond that is broken by light and is introduced into the nucleic acid, and can be used for nucleic acid sequence analysis, nucleic acid position analysis, and the like in a single cell. However, the ortho-nitrobenzyl compound used in the conventional photoreactive nucleic acid is sensitive to only light of the UV wavelength, and has a shallow depth of penetration and damage to the cell. There is a problem that is difficult to observe in the region.
또한, 종래는 광반응성 핵산을 고분자 합성 혹은 다른 분자와의 접합이 가능한 형태로 만들기 위해서는 이러한 합성 및 접합에 필요한 반응기를 광반응성 핵산을 합성한 후 별도의 과정을 거쳐 도입해야 했다.In addition, in the related art, in order to make a photoreactive nucleic acid into a form capable of polymer synthesis or conjugation with other molecules, a reactor required for such synthesis and conjugation was synthesized and then introduced through a separate process.
핵산 분석에서뿐만 아니라 핵산 기반 약물을 특정 분자에 접합시켰다가 원하는 시간 및 장소에서 빛을 조사하여 분해하여 효과를 나타내고자 할 때, 기존의 광반응성 핵산을 사용할 시에는 오르토-니트로벤질 화합물의 문제점 및 합성의 복잡성 등 동일한 문제가 존재하였다.Problems and synthesis of ortho-nitrobenzyl compounds when using existing photoreactive nucleic acids when not only in nucleic acid analysis, but also when a nucleic acid-based drug is conjugated to a specific molecule and then decomposed by irradiating light at a desired time and place The same problem existed, such as the complexity of the.
본 발명자들은 종래의 문제를 해결하기 위하여 예의 노력한 결과, 근적외선 영역의 빛에도 감응하는 쿠마린 유도체를 이용하였고, 이러한 광반응성 작용기에 고분자 합성 및 다른 분자와의 접합이 가능한 작용기를 도입하면 추가적인 단계 없이 고분자 합성 및 다른 분자와의 접합이 가능한 핵산을 제공할 수 있음을 확인하였다. 본 발명은 핵산 합성에 사용할 수 있으면서 고분자 합성 및 다른 분자와의 접합이 가능한 신규한 쿠마린 화합물을 포함하여 종래의 문제점이 개선되는 것을 확임함으로써 본 발명을 완성하였다.The present inventors used a coumarin derivative that is sensitive to light in the near-infrared region as a result of diligent efforts to solve the conventional problems, and introducing a functional group capable of polymer synthesis and conjugation with other molecules to the photoreactive functional group without additional steps. It has been confirmed that a nucleic acid capable of synthesis and conjugation with other molecules can be provided. The present invention was completed by confirming that the conventional problems were improved, including a novel coumarin compound capable of being used for nucleic acid synthesis and capable of polymer synthesis and conjugation with other molecules.
본 발명의 하나의 목적은 하기 화학식 1로 표시되는 화합물을 제공하는 것이다.One object of the present invention is to provide a compound represented by the following formula (1).
[화학식 1][Formula 1]
상기 화학식 1에서,In Chemical Formula 1,
R1은 수소 또는 C1-6알킬,R 1 is hydrogen or C 1-6 alkyl,
R2a 내지 R2b는 각각 독립적으로 C1-6알킬,R 2a to R 2b are each independently C 1-6 alkyl,
n1은 0 내지 40에서 선택되는 정수,n 1 is an integer selected from 0 to 40,
n2는 0 내지 40에서 선택되는 정수,n 2 is an integer selected from 0 to 40,
L은L is
또는 
or
상기 m은 1 내지 3에서 선택되는 정수.The m is an integer selected from 1 to 3.
본 발명의 다른 목적은 화학식 1의 화합물을 포함하는 광반응성 핵산 합성용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for synthesizing a photoreactive nucleic acid comprising a compound of Formula 1.
본 발명의 또 다른 목적은 하기 화학식 1로 표기되는 화합물과 제1핵산을 반응시켜 화학식1의 포스포르아미다이트 모이어티에 제1핵산이 결합된 제1복합체를 제조하는 제1단계 및 상기 제1복합체를 제2핵산과 접촉시켜 상보적인 서열의 핵산 이중가닥을 형성하는 제2복합체를 제조하는 제2단계를 포함하는 것인, 핵산 제조방법을 제공하는 것이다.Another object of the present invention is a first step of preparing a first complex in which a first nucleic acid is bound to a phosphoramidite moiety of Formula 1 by reacting a compound represented by the following Formula 1 with a first nucleic acid: It provides a method for producing a nucleic acid, comprising a second step of preparing a second complex to form a double-stranded nucleic acid of complementary sequence by contacting the complex with the second nucleic acid.
본 발명의 화합물은 기존의 광반응성 핵산에 사용되어온 오르토-니트로벤질 화합물은 UV 파장의 빛에만 감응하여 침투 깊이가 얕고 세포에 손상을 줄 수 있었던 문제점을 해결하고, 본 발명의 화합물이 핵산 위치 분석에 사용될 경우 UV 파장의 특성 상 초점 부피가 커 좁은 영역에서의 관찰이 어려웠던 문제점을 해결한다.The compound of the present invention solves the problem that the ortho-nitrobenzyl compound, which has been used in the conventional photoreactive nucleic acid, has a shallow penetration depth and can damage cells by responding only to UV wavelength light, and the compound of the present invention analyzes the nucleic acid position When used in the nature of the UV wavelength, it solves the problem that it was difficult to observe in a narrow area due to a large focal volume.
또한, 본 발명에서는 근적외선 영역의 빛에도 감응하는 쿠마린 유도체를 포함한 화합물을 이용하므로 침투 깊이가 깊고 세포 손상이 적게하는 효과를 제공한다. 또한, 광반응성 핵산을 고분자 중합 혹은 다른 분자와의 접합이 가능한 형태로 만들기 위해 필요했던 광반응성 핵산 합성 후의 별도의 공정이 필요하지 않으므로 경제적인 효과를 제공한다.In addition, the present invention uses a compound containing a coumarin derivative that is sensitive to light in the near-infrared region, thereby providing an effect of deep penetration and less cell damage. In addition, a separate process after synthesis of the photoreactive nucleic acid, which was necessary to make the photoreactive nucleic acid into a form capable of polymer polymerization or conjugation with other molecules, is not required, thereby providing an economical effect.
도 1은 화학식 2의 NMR분석을 나타낸 그래프이다.1 is a graph showing NMR analysis of Formula 2.
도 2는 본 발명의 화학식 2의 화합물이 연결된 복합체의 MS분석을 나타낸 그래프이다.Figure 2 is a graph showing the MS analysis of the complex of the compound of formula 2 of the present invention.
도 3은 본 발명의 화학식 2의 화합물이 연결된 복합체에 광을 조사하여 광분해한 이후 MS분석을 나타낸 그래프이다.3 is a graph showing MS analysis after photolysis by irradiating light to a complex to which the compound of Formula 2 of the present invention is connected.
도 4의 A는 화학식1의 각 작용기의 기능을 나타낸 구조식이고, B는 B는 근적외선 영역의 빛에 의한 본 발명의 화합물과 연결된 핵산의 광분해 실험 과정을 나타내는 모식도이고, C는 광분해와 형광 소광 직후의 공초점 현미경 이미지 및 D는 광분해와 형광 소광 후 새로운 형광 프로브로 핵산이 존재하는 하이드로젤을 염색한 후의 공초점 현미경 이미지이다.A of FIG. 4 is a structural formula showing the function of each functional group of Formula 1, B is a schematic diagram showing the process of photolysis of the nucleic acid linked to the compound of the present invention by light in the near-infrared region, C is immediately after photolysis and fluorescence quenching The confocal microscopy image of and D is a confocal microscopy image after staining a hydrogel containing nucleic acid with a new fluorescent probe after photolysis and fluorescence quenching.
이를 구체적으로 설명하면 다음과 같다. 한편, 본 발명에서 개시된 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 발명에서 개시된 다양한 요소들의 모든 조합이 본 발명의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 발명의 범주가 제한된다고 볼 수 없다.Specifically, it is as follows. Meanwhile, each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. In addition, the scope of the present invention is not limited by the specific descriptions described below.
상기 목적을 달성하기 위한 본 발명의 하나의 양태는, 하기 화학식 1로 표시되는 화합물을 제공한다.One aspect of the present invention for achieving the above object, provides a compound represented by the formula (1).
[화학식 1][Formula 1]
상기 화학식 1에서,In Chemical Formula 1,
R1은 수소 또는 C1-6알킬,R 1 is hydrogen or C 1-6 alkyl,
R2a 내지 R2b는 각각 독립적으로 C1-6알킬,R 2a to R 2b are each independently C 1-6 alkyl,
n1은 0 내지 40에서 선택되는 정수,n 1 is an integer selected from 0 to 40,
n2는 0 내지 40에서 선택되는 정수,n 2 is an integer selected from 0 to 40,
L은L is
또는or
상기 Y의 m은 1 내지 3에서 선택되는 정수.The m of Y is an integer selected from 1 to 3.
상기 목적을 달성하기 위한 본 발명의 다른 하나의 양태는, 하기 화학식 1의 화합물을 포함하는 광반응성 핵산 합성용 조성물을 제공한다.Another aspect of the present invention for achieving the above object is to provide a composition for synthesizing a photoreactive nucleic acid comprising a compound of Formula 1 below.
[화학식 1][Formula 1]
상기 화학식 1에서,In Chemical Formula 1,
R1은 수소 또는 C1-6알킬,R 1 is hydrogen or C 1-6 alkyl,
R2a 내지 R2b는 각각 독립적으로 C1-6알킬,R 2a to R 2b are each independently C 1-6 alkyl,
n1은 0 내지 40에서 선택되는 정수,n 1 is an integer selected from 0 to 40,
n2는 0 내지 40에서 선택되는 정수,n 2 is an integer selected from 0 to 40,
L은L is
또는or
상기 m은 1 내지 3에서 선택되는 정수.The m is an integer selected from 1 to 3.
상기 목적을 달성하기 위한 본 발명의 또 다른 하나의 양태는, 하기 화학식 1로 표기되는 화합물과 제1핵산을 반응시켜 화학식1의 포스포르아미다이트 모이어티에 제1핵산이 결합된 제1복합체를 제조하는 제1단계 및 상기 제1복합체를 제2핵산과 접촉시켜 상보적인 서열의 핵산 이중가닥을 형성하는 제2복합체를 제조하는 제2단계를 포함하는 것인, 핵산 제조방법을 제공한다.Another aspect of the present invention for achieving the above object is to react the compound represented by the following formula (1) and the first nucleic acid to form a first complex in which the first nucleic acid is bound to the phosphoramidite moiety of the formula (1). A first step of manufacturing and a second step of making a second complex of contacting the first complex with a second nucleic acid to form a nucleic acid double strand of complementary sequence are provided.
[화학식 1][Formula 1]
상기 화학식 1에서,In Chemical Formula 1,
R1은 수소 또는 C1-6알킬,R 1 is hydrogen or C 1-6 alkyl,
R2a 내지 R2b는 각각 독립적으로 C1-6알킬,R 2a to R 2b are each independently C 1-6 alkyl,
n1은 0 내지 40에서 선택되는 정수,n 1 is an integer selected from 0 to 40,
n2는 0 내지 40에서 선택되는 정수,n 2 is an integer selected from 0 to 40,
L은L is
또는 
or
상기 m은 1 내지 3에서 선택되는 정수.The m is an integer selected from 1 to 3.
구체적으로, 상기 화합물은 핵산 합성, 핵산 분리 및 복합체 형성 용도로 사용되는 것일 수 있고, 기존의 광반응성 핵산에 사용되어온 오르토-니트로벤질 화합물은 UV 파장의 빛에만 감응하여 침투 깊이가 얕고 세포에 손상을 줄 수 있었던 문제점을 해결하고, 본 발명의 화합물이 핵산 위치 분석에 사용될 경우 UV 파장의 특성 상 초점 부피가 커 좁은 영역에서의 관찰이 어려웠던 문제점을 해결하고, UV 뿐만 아니라 근적외선 영역의 빛에도 감응하는 쿠마린 유도체를 포함한 화합물을 이용하므로 침투 깊이가 깊고 세포 손상이 적게하는 효과를 제공 및 광반응성 핵산을 고분자 중합 혹은 다른 분자와의 접합이 가능한 형태로 만들기 위해 필요했던 광반응성 핵산 합성 후의 별도의 공정이 필요하지 않으므로 경제적인 효과를 제공할 수 있다.Specifically, the compound may be used for nucleic acid synthesis, nucleic acid separation and complex formation, and the ortho-nitrobenzyl compound that has been used in the existing photoreactive nucleic acid is sensitive to UV light only and has a shallow penetration depth and damages cells. When the compound of the present invention is used for nucleic acid position analysis, it solves a problem that it is difficult to observe in a narrow region due to the large focus volume due to the nature of the UV wavelength, and is sensitive to light in the near infrared region as well as UV. Since it uses a compound containing a coumarin derivative, it provides an effect of deep penetration and less cell damage, and a separate process after photoreactive nucleic acid synthesis, which was necessary to polymerize or polymerize the photoreactive nucleic acid to other molecules. Since this is not necessary, it can provide an economic effect.
본 발명의 용어, "핵산 합성(Solid-phase oligonucleotide synthesis)"은 뉴클레오티드 즉, 핵산의 구성 단위. 뉴클레오시드 당부분의 인산에스테르가 각각 상보적인 서열 상호작용에 의해 합성되는 것을 의미한다.The term "nucleic acid synthesis (Solid-phase oligonucleotide synthesis)" of the present invention is a nucleotide, that is, a structural unit of a nucleic acid. It means that the phosphate ester of the nucleoside sugar moiety is synthesized by complementary sequence interaction, respectively.
본 발명의 용어, "복합체 형성"은 본 발명의 제1핵산과 제2핵산이 상보적인 서열 작용에 의해 핵산 이중가닥을 형성하는 것을 의미할 수 있고, 상기 제2핵산은 제1핵산의 일부 또는 전부와 상보적인 서열을 갖는 단일가닥 핵산 또는 A, G, C 및 T 핵산 단량체의 혼합물인 것일 수 있고, 상기 제2핵산은 제1핵산과 상보적인 서열 또는 그와 유사한 서열을 포함하여 핵산 사이의 상호작용에 따라 제2복합체를 의미할 수 있다.The term "complex formation" of the present invention may mean that the first nucleic acid and the second nucleic acid of the present invention form a double-stranded nucleic acid by complementary sequence action, wherein the second nucleic acid is a part of the first nucleic acid or It may be a single-stranded nucleic acid having a sequence complementary to all or a mixture of A, G, C and T nucleic acid monomers, wherein the second nucleic acid is between the nucleic acid including a sequence complementary to the first nucleic acid or a sequence similar thereto. According to the interaction, it may mean the second complex.
본 발명의 용어, "핵산 분리"는 핵산과 다른 화합물이 결합되어 있는 복합체로부터 핵산과 다른 화합물이 분리되는 것을 의미한다.The term "nucleic acid separation" of the present invention means that a nucleic acid and another compound are separated from a complex in which a nucleic acid and other compounds are bound.
구체적으로, 본 발명의 제3복합체에 광을 조사하면 화학식 1로 표기되는 화합물 및 조성물과 분리되어 핵산만을 얻을 수 있는 것을 말할 수 있다.Specifically, when the third complex of the present invention is irradiated with light, it can be said that only the nucleic acid can be obtained by being separated from the compound and composition represented by Chemical Formula 1.
상기 화합물은 하기 화학식 2, 화학식 3, 또는 화학식 4로 표시되는 것일 수 있다.The compound may be represented by the following Chemical Formula 2, Chemical Formula 3, or Chemical Formula 4.
[화학식 2][Formula 2]
[화학식 3][Formula 3]
[화학식 4][Formula 4]
상기 화학식 2 내지 화학식 4 에서,In Chemical Formulas 2 to 4,
R1은 수소 또는 C1-6알킬,R 1 is hydrogen or C 1-6 alkyl,
R2a 내지 R2b는 각각 독립적으로 C1-6알킬,R 2a to R 2b are each independently C 1-6 alkyl,
n은 0 내지 40에서 선택되는 정수,n is an integer selected from 0 to 40,
m은 1 내지 3에서 선택되는 정수.m is an integer selected from 1 to 3.
본 발명의 용어, "쿠마린"은 헤테로고리계열에 속하는 유기화합물로서 외관상 무색 결정인데, 통카콩(tonka bean)의 방향성분으로 알려져 있어 향료로 사용되고, 벤조-α피론이라고도 하며, o-옥시신남산의 락톤에 해당한다. 화학식 C9H6O2. 통카콩의 방향 성분으로 미량이지만 각종 식물, 특히 열매에 함유되어 있다. 무색의 결정이며, 분자량 146.15, 녹는점 71℃ 끓는점 301.7℃ 비중 0.935이다. 찬물에는 녹기 어렵지만 뜨거운 물에는 녹는다. 살리실알데하이드와 아세트산무수물을 아세트산칼륨의 무수물 존재하에서 축합하면 얻고, 향료로 사용되며 치환체에도 향료가 되는 것이 많은 것을 의미한다.The term "coumarin" of the present invention is an organic compound belonging to the heterocyclic family, and is a colorless crystal in appearance. It is known as an aromatic component of tonka bean and is used as a fragrance. Also called benzo-αpyrone, o-oxycinnamic acid Corresponds to lactone. Formula C 9 H 6 O 2 . It is a very small amount of aroma component of tonka bean, but it is contained in various plants, especially fruits. It is a colorless crystal and has a molecular weight of 146.15, a melting point of 71 ° C, a boiling point of 301.7 ° C, and a specific gravity of 0.935. It is difficult to dissolve in cold water, but dissolves in hot water. It means that salicylaldehyde and acetic anhydride are obtained by condensation in the presence of anhydrous potassium acetate, which is used as a fragrance and is often used as a fragrance for a substituent.
구체적으로, 상기 쿠마린은 7-하이드록시-4-메틸쿠마린을 포함하는 것일 수 있다.Specifically, the coumarin may be one containing 7-hydroxy-4-methylcoumarin.
예컨대, 본 발명의 화합물은 7-하이드록시-4-메틸쿠마린을 모핵에 포함하는 화합물일 수 있다.For example, the compound of the present invention may be a compound containing 7-hydroxy-4-methylcoumarin in the parent nucleus.
본 발명의 화합물 및 조성물은 쿠마린을 포함하고 쿠마린은 광반응성이 우수하여 높은 효율로 이광자 현상 즉, 낮은 에너지의 근적외선으로 흡수하여 활성화가 가능할 수 있다.The compounds and compositions of the present invention include coumarin, and coumarin is excellent in photoreactivity, so it can be activated by absorbing it in a near-infrared light with a high efficiency, i.
본 발명의 화합물 및 조성물에 포함된 비닐기가 고분자 네트워크겔에 포함된 고분자와 결합될 수 있고, 고분자에 고정된 제3복합체는 광을 조사하기 용이하게 고정될 수 있다.The vinyl group included in the compounds and compositions of the present invention can be combined with the polymer contained in the polymer network gel, and the third complex fixed to the polymer can be easily fixed to irradiate light.
상기 쿠마린은 광분해성인 것일 수 있다.The coumarin may be photodegradable.
*98본 발명의 용어, "광분해"는 광에 반응하여 빛을 흡수함으로써 일어나는 분리로 빛을 흡수한 물질이 분리하는 직접광분해와 빛을 흡수하여 생긴 여기광증감제로부터의 에너지 이동이나 화학반응에 의해 반응물질이 분리하는 광증감분리가 있고, 한 분자가 빛을 흡수하여 두 가지 이상의 성분으로 분리되는 것을 의미한다.* 98 The term "photolysis" of the present invention is a separation that occurs by absorbing light in response to light, and is used for direct photolysis that is absorbed by light-absorbing substances and energy transfer or chemical reaction from excitation light-sensitizers generated by absorbing light. There is a photosensitive separation separated by a reactant, and it means that one molecule absorbs light and is separated into two or more components.
본 발명의 화합물은 광반응성 물질로 쿠마린 연결체를 포함하여 쿠마린은 빛에너지를 화학에너지로 전환시키며, 광화학 작용에 의해 광반응으로 더 적은 분자량을 가지는 물질로 변환되는 광분해 될 수 있다.The compound of the present invention includes a coumarin linker as a photoreactive material, and coumarin converts light energy into chemical energy, and can be photodegraded to be converted into a material having a lower molecular weight by photoreaction by photochemical action.
구체적인 일 실시예에서는, 본 발명의 화합물이 광반응 하여 핵산을 제3복합체로부터 분리되어 핵산만을 얻는 것을 확인하였다.In a specific embodiment, it was confirmed that the compound of the present invention was photoreacted to separate the nucleic acid from the third complex to obtain only the nucleic acid.
상기 조성물은 화학식 1의 화합물에 포함된 비닐기를 통해 고분자 네트워크겔과 결합하는 것일 수 있다.The composition may be to combine with a polymer network gel through a vinyl group included in the compound of formula (1).
본 발명의 용어, "비닐기(vinyl group)"는 CH2=CH-로 표시되는 작용기로 염화비닐 등의 고분자화합물을 만드는 데 사용되며, 이중결합을 가지기 때문에 이 기를 가진 화합물은 반응성이 풍부하며 합성하여 고분자화합물을 잘 만드는 것을 의미한다.The term "vinyl group" of the present invention is a functional group represented by CH 2 = CH- and is used to make a polymer compound such as vinyl chloride, and since it has a double bond, the compound having this group is rich in reactivity. It means to make a high molecular compound by synthesis.
본 발명의 화합물은 비닐기를 포함하여 고분자 네트워크로겔과 중합 시 공중합될 수 있다. 본 발명의 화합물이 고정되는 고분자 네트워크겔은 핵산을 합성 및 분리할 수 있게 하는 매개체가 될 수 있다. 본 발명의 화합물은 비닐기를 포함하여 고분자 네트워크에 고정 또는 비닐기와의 반응으로 화학 결합을 형성할 수 있는 물질과 접합될 수 있다.The compound of the present invention may be copolymerized when polymerized with a polymer network gel including a vinyl group. The polymer network gel to which the compound of the present invention is immobilized may be a medium capable of synthesizing and isolating nucleic acids. The compound of the present invention may be fixed to a polymer network including a vinyl group or conjugated with a material capable of forming a chemical bond by reaction with a vinyl group.
상기 고분자 네트워크기겔은 비닐기와 반응 가능한 물질 일 수 있다.The polymer network gel may be a material capable of reacting with a vinyl group.
상기 조성물은 화학식 1의 화합물에 포함된 포스포르아미다이트 모이어티를 통해 핵산과 합성하는 것일 수 있다.The composition may be synthesized with a nucleic acid through a phosphoramidite moiety included in the compound of Formula 1.
본 발명의 용어, "포스포르아미다이트 모이어티(phosphoramidite moiety)"는 본 발명의 화학식 1에서 비닐기를 제외한 하기 화학식 1-1로 표시되는 부분을 나타낼 수 있고, 이 부분을 통해 핵산과 결합을 형성할 수 있다:The term "phosphoramidite moiety (phosphoramidite moiety)" of the present invention may indicate a portion represented by the following formula 1-1 except for the vinyl group in the formula 1 of the present invention, through this portion to bind to the nucleic acid Can form:
[화학식 1-1][Formula 1-1]
화학식 1의 화합물은 양끝에 비닐기 및 포함된 포스포르아미다이트 모이어티를 포함하여 비닐기에서는 고분자 네트워크겔과 결합 될 수 있고, 포스포르아미다이트 모이어티 부분에서는 제1핵산과 결합하고 제1핵산과 제2핵산이 상보적인 서열의 핵산 이중가닥을 형성하여 제2복합체를 형성할 수 있고, 본 발명의 제3복합체로 표기되는 고분자 네트워크겔-화합물-핵산의 형태로 복합체를 형성할 수 있다.The compound of Formula 1 may include a vinyl group at both ends and an embedded phosphoramidite moiety, and may be combined with a polymer network gel in the vinyl group, and binds to the first nucleic acid in the phosphoramidite moiety portion. The first nucleic acid and the second nucleic acid can form a double-stranded nucleic acid having a complementary sequence to form a second complex, and can form a complex in the form of a polymer network gel-compound-nucleic acid represented by the third complex of the present invention. have.
상기 조성물은 적외선 조사시 화학식 1의 화합물에 포함된 쿠마린에 의해 이광자 흡수를 통해 합성된 핵산을 분리하는 것일 수 있다.The composition may be to separate nucleic acids synthesized through diphoton absorption by coumarin contained in the compound of Formula 1 when irradiated with infrared rays.
구체적으로, 본 발명의 조성물은 쿠마린을 포함하여 적외선에 광반응 할 수 있고, 본 발명의 조성물과 결합된 핵산은 적외선 파장의 광에 반응하여 분리될 수 있다.Specifically, the composition of the present invention may be photoreactive to infrared light including coumarin, and the nucleic acid combined with the composition of the present invention may be separated by reacting to light having an infrared wavelength.
하기 화학식 1로 표기되는 화합물과 제1핵산을 반응시켜 화학식1의 포스포르아미다이트 모이어티에 제1핵산이 결합된 제1복합체를 제조하는 제1단계 및 상기 제1복합체를 제2핵산과 접촉시켜 상보적인 서열의 핵산 이중가닥을 형성하는 제2복합체를 제조하는 제2단계를 포함할 수 있다.A first step of preparing a first complex in which a first nucleic acid is bonded to a phosphoramidite moiety of Formula 1 by reacting a compound represented by the following Chemical Formula 1 with a first nucleic acid, and contacting the first complex with a second nucleic acid And a second step of preparing a second complex to form a double-stranded nucleic acid of complementary sequence.
또한, 상기 제2핵산은 제1핵산의 일부 또는 전부와 상보적인 서열을 갖는 단일가닥 핵산 또는 A, G, C 및 T 핵산 단량체의 혼합물인 것일 수 있다.In addition, the second nucleic acid may be a single-stranded nucleic acid having a sequence complementary to some or all of the first nucleic acid or a mixture of A, G, C and T nucleic acid monomers.
또한, 상기 제2단계 이전 또는 이후에 화학식1의 비닐기에 고분자 네트워크겔을 결합시켜 제3복합체를 형성하는 제3단계를 추가로 포함하는 것일 수 있다.In addition, the third step of forming a third complex by bonding the polymer network gel to the vinyl group of Formula 1 before or after the second step may be further included.
또한, 상기 제3 단계 이후 상기 상기 화합물에 광을 조사하여 핵산을 분리하는 단계를 더 포함하는 것일 수 있다.In addition, after the third step, it may further include the step of isolating the nucleic acid by irradiating the compound with light.
구체적으로, 상기 제1복합체는 화학식 1-제1핵산으로 표기될 수 있고 상기 제2복합체는 화학식 1-제2핵산-제2핵산 형태로 형성할 수 있고, 제3복합체는 고분자 네트워크겔-화학식1-제1핵산-제2핵산형태로 형성될 수 있다 광을 조사하여 화학식1에 포함된 쿠마린이 광반응하여 고분자 네트워크겔-화학식 1의 형태 및 제1핵산-제2핵산 형태로 분리될 수 있어 핵산을 수득할 수 있다.Specifically, the first complex may be represented by Formula 1-first nucleic acid, the second complex may be formed in the form of Formula 1-second nucleic acid-second nucleic acid, and the third complex is a polymer network gel-formula It can be formed in the form of 1-first nucleic acid-second nucleic acid. The coumarin contained in Chemical Formula 1 is photoreacted by irradiating light to be separated into a polymer network gel-form of Formula 1 and a first nucleic acid-second form of nucleic acid. To obtain a nucleic acid.
상기 광은 자외선, 가시광선 또는 적외선인 것일 수 있다.The light may be ultraviolet, visible or infrared.
본 발명의 용어, "자외선(UV)"은 자외선은 파장 범위 10~400 nm(에너지 범위 3 eV ~ 124 eV)인 빛으로서, 파장이 가시광보다 더 짧고 엑스선보다는 더 긴 전자기파이며, 유리는 거의 투과할 수 없고, 공기도 잘 통과하지 못한다. 진동수가 큰 고에너지파로 생명체의 분자들을 부분적으로 파괴하거나 분리할 정도로 화학 작용을 강하게 일으키는 것을 의미한다.The term “ultraviolet rays (UV)” of the present invention refers to ultraviolet light having a wavelength ranging from 10 to 400 nm (energy range 3 eV to 124 eV), which is shorter than visible light and longer than X-rays, and is almost transparent to glass. It cannot, and the air does not pass well. It is a high-energy wave with a high frequency, which means that the chemical action is strong enough to partially destroy or separate molecules in life.
본 발명의 용어, "가시광선"은 눈으로 지각되는 파장 범위를 가진 빛. 물리적인 빛은 눈에 색채로서 지각되는 범위의 파장 한계 내에 있는 스펙트럼이며, 대략 380~780nm(nanometer) 범위의 파장을 가진 전자파를 의미한다.The term "visible light" in the present invention is light having a wavelength range perceived by the eye. Physical light is a spectrum within the wavelength limit of the range perceived as color to the eye, and refers to electromagnetic waves having a wavelength in the range of approximately 380 to 780 nm (nanometer).
본 발명의 용어, "적외선"은 가시광선보다 파장이 길며, 0.75μm에서 1mm 범위에 속하는 전자기파. 햇빛이나 백열된 물체로부터 방출되는 빛을 스펙트럼으로 분산시켜 보면 적색스펙트럼의 끝보다 더 바깥쪽에 있으므로 적외선이라 한다. 파장 0.75 ~3μm의 적외선을 근적외선, 3-25μm의 것을 단순히 적외선이라 하며, 25μm 이상의 것을 원적외선이라 한다. 가시광선이나 자외선에 비해 강한 열작용을 가지고 있는 것이 특징이며, 이 때문에 열선이라고도 하는 것을 의미한다.The term "infrared" of the present invention is an electromagnetic wave having a longer wavelength than visible light and falling in a range of 0.75 μm to 1 mm. Dispersion of light emitted from sunlight or incandescent objects into the spectrum is called infrared because it is located farther than the end of the red spectrum. Infrared rays with a wavelength of 0.75 to 3 μm are called near infrared, those with 3-25 μm are simply called infrared, and those with a wavelength of 25 μm or more are called far infrared. It is characterized by having a stronger thermal action than visible light or ultraviolet light, and for this reason, it is also referred to as heat rays.
본 발명의 화합물은 UV 뿐만 아니라 근적외선 영역에서도 감응하는 쿠마린을 포함하여 자외선(UV)에 만 반응하여 침투 깊이가 얕고 세포에 손상을 주는 문제점 및 핵산 위치 분석에 사용시 상 초첨 부피가 커 좁은 영역에 관찰이 어려웠던 문제점을 해결할 수 있다.The compounds of the present invention respond to UV (UV) only, including coumarins that respond not only in the UV but also in the near-infrared region, resulting in shallow penetration depth and damage to the cells. You can solve this difficult problem.
구체적인 일 실시예에서는, 본 발명의 화합물을 이용하여 UV뿐만아니라 근적외선에서 반응하므로 UV에만 반응하여 침투 깊이가 얕고 세포에 손상을 줄 수 있었던 문제점 및 부피가 커 좁은 영역에서의 관찰이 어려웠던 종래 문제점을 해결하고, 또한, 광반응성 핵산을 고분자 중합 혹은 다른 분자와의 접합이 가능한 형태로 만들기 위해 필요했던 광반응성 핵산 합성 후의 별도의 공정이 필요하지 않으므로 경제적인 효과를 확인하였다.In a specific embodiment, since the compound of the present invention reacts in the UV as well as near-infrared rays, it reacts only to UV, causing a problem that shallow penetration depth and damage to cells and difficulty in observing in a small volume are difficult. In addition, an economic effect was confirmed because a separate process after photoreactive nucleic acid synthesis, which was necessary to make the photoreactive nucleic acid polymerizable or conjugated with other molecules, is not required.
이하, 실시예를 통하여 본 발명을 보다 상세히 설명하고자 한다. 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited by these examples.
실시예 1: 쿠마린을 포함한 광반응성 화합물의 제조Example 1: Preparation of photoreactive compounds including coumarin
쿠마린을 포함한 광반응성 화합물의(화학식 2)의 제조는 하기 반응식 1에 따라 제조하였다.Preparation of a photoreactive compound containing coumarin (Formula 2) was prepared according to Scheme 1 below.
[반응식 1][Scheme 1]
a단계: 먼저, 화합물 2를 제조(반응식 1의 2로 표기되는 화합물 2)하였다. 7-하이드록시-4-메틸쿠마린 (0.30 g, 1.70 mmol) 을 아세톤 (18 mL)에 용해시키고 K2CO3 (0.259 g, 1.87 mmol)와 크라운에테르 (0.450 g, 1.70 mmol)를 가하였다. 리플럭스 장치에서 1시간 동안 교반한 후 2-터트부틸록시카보닐 아미노에틸브로마이드 (0.42 g, 1.87 mmol)를 가하였다. 리플럭스 장치에서 18시간 동안 교반한 후 여과한 뒤 농축시켰다. 실리카 겔 플래쉬 컬럼 크로마토그래피로 정제시켜 흰색 고체의 화합물 2를 수득하였다. (0.483 g, 88% 수율)Step a: First, compound 2 was prepared (compound 2 represented by 2 in scheme 1). 7-hydroxy-4-methylcoumarin (0.30 g, 1.70 mmol) was dissolved in acetone (18 mL) and K 2 CO 3 (0.259 g, 1.87 mmol) and crown ether (0.450 g, 1.70 mmol) were added. After stirring for 1 hour in a reflux apparatus, 2-tertbutyloxycarbonyl aminoethyl bromide (0.42 g, 1.87 mmol) was added. After stirring for 18 hours in a reflux device, it was filtered and concentrated. Purification by silica gel flash column chromatography gave compound 2 as a white solid. (0.483 g, 88% yield)
b단계: 그 다음으로, 화합물 3을 제조(반응식 1의 3으로 표기되는 화합물 3을 제조하는 b반응)하였다.Step b: Next, compound 3 was prepared (b reaction to produce compound 3 represented by 3 in scheme 1).
상기 화합물 2 (0.30 g, 0.94 mmol)를 디메틸포름아미드 (1 mL)에 용해시키고 교반시키면서 디메틸포름아미드 디메틸 아세탈(0.25 mL, 1.8 mmol)을 가하였다. 리플럭스 장치에서 14시간 동안 교반한 후 상온으로 냉각시켰다. 그런 다음 포화 NaHCO3 용액을 가하고 CH2Cl2로 추출하였다. 얻어진 유기층은 무수 MgSO4로 건조시키고 여과한 뒤 감압 하에 농축시켜 crude 화합물 3을 수득하였다. 별도의 정제과정 없이 다음 반응에 사용하였다.Compound 2 (0.30 g, 0.94 mmol) was dissolved in dimethylformamide (1 mL) and dimethylformamide dimethyl acetal (0.25 mL, 1.8 mmol) was added with stirring. The mixture was stirred for 14 hours in a reflux device and then cooled to room temperature. Then saturated NaHCO 3 solution was added and extracted with CH 2 Cl 2 . The obtained organic layer was dried over anhydrous MgSO 4 , filtered and concentrated under reduced pressure to obtain crude compound 3. It was used for the next reaction without any purification.
c단계: 화합물 4를 제조(반응식 1의 4로 표기되는 화합물 4를 제조하는 c반응)하였다.Step c: Compound 4 was prepared (c-reaction to prepare compound 4 represented by 4 in Scheme 1).
상기 crude 화합물 3을 물 (4 mL)과 THF (4 mL)에 용해시키고 NaIO4 (0.60 g, 2.8 mmol)을 가하였다. 상온에서 2시간 동안 교반한 후 여과한 뒤 감압하여 농축하였다. 그런 다음 포화 NaHCO3 용액을 가하고 CH2Cl2로 추출하였다. 얻어진 유기층은 무수 MgSO4로 건조시키고 여과한 뒤 감압 하에 농축시켜 crude 화합물 4를 수득하였다. 별도의 정제과정 없이 다음 반응에 사용하였다.The crude compound 3 was dissolved in water (4 mL) and THF (4 mL) and NaIO 4 (0.60 g, 2.8 mmol) was added. After stirring at room temperature for 2 hours, filtered and concentrated under reduced pressure. Then saturated NaHCO 3 solution was added and extracted with CH 2 Cl 2 . The obtained organic layer was dried over anhydrous MgSO 4 , filtered and concentrated under reduced pressure to obtain crude compound 4. It was used for the next reaction without a separate purification process.
d단계: 화합물 5를 제조(반응식 1의 5로 표기되는 화합물 5를 제조하는 d반응)하였다.Step d: Compound 5 was prepared (d reaction to produce compound 5 represented by 5 in Scheme 1).
상기 crude 화합물 4를 무수 THF (9 mL)에 용해시키고 0℃로 냉각시킨 뒤 NaBH4 (0.071 g, 1.9 mmol)을 가하였다. 상온에서 2시간 동안 교반한 후 포화 NaHCO3 용액을 가하고 CH2Cl2로 추출하였다. 얻어진 유기층은 무수 MgSO4로 건조시키고 여과한 뒤 감압 하에 농축시켜 crude 화합물 4를 수득하였다. 실리카 겔 컬럼 크로마토그래피로 정제시켜 연한 노란색 고체의 화합물 5를 수득하였다. (0.14 g, 46% 수율)The crude compound 4 was dissolved in anhydrous THF (9 mL), cooled to 0 ° C., and NaBH 4 (0.071 g, 1.9 mmol) was added. After stirring at room temperature for 2 hours, saturated NaHCO 3 solution was added and extracted with CH 2 Cl 2 . The obtained organic layer was dried over anhydrous MgSO 4 , filtered and concentrated under reduced pressure to obtain crude compound 4. Purification by silica gel column chromatography gave compound 5 as a light yellow solid. (0.14 g, 46% yield)
e단계: 화합물 6을 제조(반응식 1의 6으로 표기되는 화합물 6을 제조하는 e반응)하였다.Step e: Compound 6 was prepared (e-reaction to prepare compound 6 represented by 6 in Scheme 1).
상기 화합물 5(0.10 g, 0.30 mmol)를 디클로로메테인 (10 mL)에 용해시킨 뒤 트리플루오로아세트산 (1.6 mL, 21 mmol)을 천천히 가하였다. 상온에서 2시간 동안 교반한 뒤 TFA를 증발시키고 디에틸에테르를 가해 침전시켰다. 감압 하에 농축시켜 화합물 6을 수득하였다. (0.098 g, 94% 수율)The compound 5 (0.10 g, 0.30 mmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (1.6 mL, 21 mmol) was slowly added. After stirring at room temperature for 2 hours, TFA was evaporated and diethyl ether was added to precipitate. Concentration under reduced pressure gave compound 6. (0.098 g, 94% yield)
f단계: 화합물 7의 제조(반응식 1의 7로 표기되는 화합물 7을 제조하는 f반응)Step f: Preparation of compound 7 (f reaction to prepare compound 7 represented by 7 in scheme 1)
상기 화합물 6(0.44 g, 1.3 mmol)을 무수 아세토니트릴 (6.6 mL)에 용해시킨 뒤 트리에틸아민 (0.22 mL, 1.6 mmol)과 숙신이미딜 메타크릴레이트 (0.27 g, 1.5 mmol)를 가하였다. 0℃로 냉각시킨 뒤 상온에서 16시간 동안 교반하였다. 그런 다음 0.01 M HCl을 가하고 에틸아세테이트로 추출하였다. 유기층은 포화 NaHCO3 용액으로 씻은 후 무수 MgSO4로 건조시키고 여과한 뒤 감압 하에 농축시켰다. 실리카 겔 플래쉬 컬럼 크로마토그래피로 정제시켜 연한 노란색 고체의 화합물 7을 수득하였다. (0.23 g, 59% 수율)The compound 6 (0.44 g, 1.3 mmol) was dissolved in anhydrous acetonitrile (6.6 mL), and then triethylamine (0.22 mL, 1.6 mmol) and succinimidyl methacrylate (0.27 g, 1.5 mmol) were added. After cooling to 0 ° C., the mixture was stirred at room temperature for 16 hours. Then 0.01 M HCl was added and extracted with ethyl acetate. The organic layer was washed with saturated NaHCO 3 solution, dried over anhydrous MgSO 4 , filtered and concentrated under reduced pressure. Purification by silica gel flash column chromatography gave compound 7 as a light yellow solid. (0.23 g, 59% yield)
g단계: 화학식 2을 제조(반응식 1의 8로 표기되는 화학식 2을 제조하는 g반응)하였다.Step g: Formula 2 was prepared (g reaction to prepare Formula 2 represented by 8 in Scheme 1).
상기 화합물 7(0.2 g, 0.66 mmol)을 무수 아세토니트릴 (3.5 mL)에 용해시킨 뒤 1H-트리아졸 (1.5 mL, 0.72 mmol)과 2-시아노에틸 N,N,N',N'-테트라이소프로필포스포르디아미디트 (0.42 mL, 1.3 mmol)를 천천히 가하였다. 상온에서 16시간 동안 교반한 후 여과한 뒤 감압하여 농축하였다. 그런 다음 에틸아세테이트를 가하고 포화 NaHCO3 용액으로 씻어주었다. 포화 NaHCO3 용액은 다시 에틸아세테이트로 추출하였다. 유기층은 포화 염화나트륨 용액으로 씻은 뒤 무수 MgSO4로 건조하여 여과한 뒤 감압 하에 농축시켰다. 실리카 겔 컬럼 크로마토그래피로 정제시켜 흰색 고체의 쿠마린을 포함한 광반응성 화합물(화학식 2)을 수득하였다. (0.19 g, 57% 수율)The compound 7 (0.2 g, 0.66 mmol) was dissolved in anhydrous acetonitrile (3.5 mL), followed by 1H-triazole (1.5 mL, 0.72 mmol) and 2-cyanoethyl N, N, N ', N'-tetra Isopropylphosphodiamidite (0.42 mL, 1.3 mmol) was added slowly. After stirring at room temperature for 16 hours, filtered and concentrated under reduced pressure. Then ethyl acetate was added and washed with saturated NaHCO 3 solution. The saturated NaHCO 3 solution was extracted again with ethyl acetate. The organic layer was washed with saturated sodium chloride solution, dried over anhydrous MgSO 4 , filtered, and concentrated under reduced pressure. Purification by silica gel column chromatography afforded a photoreactive compound (Formula 2) containing white solid coumarin. (0.19 g, 57% yield)
실시예 2: 핵산 합성 및 분리방법Example 2: nucleic acid synthesis and separation method
상기 실시예 1로 제조된 화학식 2에 제1핵산을 반응시켜 화학식1의 포스포르아미다이트 모이어티에 제1핵산이 결합된 제1복합체를 제조하였고, 제1복합체에 생체시료에 포함된 제2핵산을 접촉시켜 상보적인 서열의 핵산 이중가닥을 형성하는 제2복합체를 제조하여 핵산을 합성하였고, 화학식2의 비닐기에 고분자 네트워크겔을 결합시켜 제3복합체를 형성하였다.A first complex in which the first nucleic acid is bound to the phosphoramidite moiety of Formula 1 was prepared by reacting the first nucleic acid with Formula 2 prepared in Example 1, and the second compound contained in the biological sample in the first complex. Nucleic acid was synthesized by contacting the nucleic acid to form a nucleic acid double strand having a complementary sequence, and a polymer network gel was bonded to a vinyl group of Formula 2 to form a third complex.
제3복합체에 근적외선 780nm파장의 이광자레이저를 25%출력으로 조사하여 화학식 2의 쿠마린으로부터 광반응을 통해 핵산이 분리되어 핵산을 얻을 수 있다.The third complex is irradiated with a near-infrared 780nm wavelength biphoton laser at 25% output, and nucleic acid is separated from the coumarin of Formula 2 through photoreaction to obtain nucleic acid.
실험예 1: NMR 분석 실험Experimental Example 1: NMR analysis experiment
상기 실시예 1로 제조된 화학식 2를 분석하기 위해 NMR분석 실험을 하였다.In order to analyze Formula 2 prepared in Example 1, an NMR analysis experiment was performed.
도 1에 나타난 바와 같이, NMR분석 결과 화학식 2와 의 n 및 m이 정수 1인 것을 확일 할 수 있었고, R1은 메틸 R2a 및 R2b는 이소프로필인 것을 확인할 수 있었다. 또한, 화학식 2의 말미에는 비닐기를 포함하여 고분자와 결합되는 작용기를 확인하였고, 쿠마린을 포함하여 광반응 할 수 있는 작용기를 확인하고, 포스포르아미다이트 모이어티를 포함하여 핵산과 결합되는 부분을 확인할 수 있었다.As shown in FIG. 1, as a result of NMR analysis, it can be confirmed that n and m in Formula 2 are integers 1, and R 1 is methyl R 2a and R 2b is isopropyl. In addition, at the end of Formula 2, a functional group that is bonded to a polymer including a vinyl group was identified, a functional group capable of photoreaction including coumarin, and a portion bound to a nucleic acid, including a phosphoramidite moiety. I could confirm.
실험예 2: MS 분석 실험Experimental Example 2: MS analysis experiment
상기 실시예 2에서 제조한 복합체를 자외선을 조사하기 전 및 조사한 후에 MALDI-TOF를 이용하여 질량을 분석하였다.The composite prepared in Example 2 was analyzed for mass using MALDI-TOF before and after ultraviolet irradiation.
표 1 및 도 2 내지 도 3에서 나타난 바와 같이, 자외선을 조사하기 전의 복합체의 경우 도2 와 같이 본 발명의 화합물 화학식2와 합성된 핵산의 질량이 관찰되었다. 자외선을 조사한 이후 광분해된 복합체의 경우, 도 3과 같이 화학식2의 화합물을 제외한 온전한 핵산의 질량만이 관찰되었다. 이를 통해 자외선 영역의 빛을 처리하였을 때 복합체는 본 발명의 화합물을 포함하여 광에 반응하여 분리되는 것을 확인하였고 효과적으로 핵산을 분리하는 것을 확인하였다.As shown in Table 1 and FIGS. 2 to 3, in the case of the complex before irradiation with ultraviolet light, the mass of the nucleic acid synthesized with the compound of Formula 2 of the present invention was observed as shown in FIG. In the case of the photodegraded complex after irradiation with ultraviolet rays, as shown in FIG. 3, only the mass of the intact nucleic acid except for the compound of Formula 2 was observed. Through this, when the light in the ultraviolet region was treated, it was confirmed that the complex was separated in response to light, including the compound of the present invention, and it was confirmed that the nucleic acid was effectively separated.
실험예 3: 이광자 분해 실험Experimental Example 3: Two-photon decomposition experiment
화학식 2와 연결된 poly(dT)20 핵산을 아크릴아마이드 단량체와 APS 개시제를 이용해 공중합하여 고분자 네트워크로 구성된 겔을 형성한 뒤 형광이 달린 poly(dA) 프로브를 이용하여 겔에 존재하는 poly(dT)를 염색하였다. 해당 겔의 특정 부분에는 (도 4C의 ★) 근적외선 780 nm 파장의 이광자 레이저를 조사하여 화학식 2의 광분해를 유도하였고 겔의 다른 부분에는 (도 4C의 #) 488 nm 파장의 단광자 레이저를 이용하여 poly(dA) 프로브의 형광을 소광시켰다. 도 4B의 모식도에서 나타난 바와 같이, 가열을 통해 기존의 형광 프로브를 제거하고 새로운 형광 프로브를 넣어주었을 때 형광반응을 관찰하였다.The poly (dT) 20 nucleic acid linked to Formula 2 is copolymerized with an acrylamide monomer and an APS initiator to form a gel composed of a polymer network, and then the poly (dT) present in the gel is used using a fluorescent poly (dA) probe. Dyed. A specific part of the gel (★ in FIG. 4C) was irradiated with a near-infrared 780 nm wavelength photon laser to induce photolysis of Formula 2, and the other part of the gel (# in FIG. 4C) was used with a single photon laser of 488 nm wavelength. Fluorescence of the poly (dA) probe was quenched. As shown in the schematic diagram of FIG. 4B, the fluorescence reaction was observed when the existing fluorescent probe was removed through heating and a new fluorescent probe was added.
도 4에서 나타난 바와 같이, 본 발명의 복합체를 나타낸 도 4의 A는 도 4의 B의 모식도와 같이 두가지 파장을 조사하여 광분해 실험을 하였다. 도 4의 C 및 D와 같이 # 부분은 형광만을 488nm파장의 단광자 레이저로 퀜칭하여 소광시킬 수 있고 ★ 부분은 780nm파장의 이광자 레이저를 조사하여 복합체에서 핵산만 분리되어 광분해 될 수 있음을 확인하였다. 따라서, 도 4의 C는 형광을 퀜칭했지만 새로운 probe를 부착할 경우 형광을 관찰할 수 있고, D는 근적외선 파장의 이광자레이저를 조사하면 광분해가 일어나 복합체에서 핵산만 분리되어 ★ 부분에만 형광 신호가 존재하지 않음을 확인함으로, 근적외선에서 감응하여 광분해되고 핵산이 분리된 것을 확인하였다.As shown in FIG. 4, A of FIG. 4 showing the composite of the present invention was subjected to a photolysis experiment by irradiating two wavelengths as shown in the schematic diagram of B of FIG. 4. As shown in C and D of FIG. 4, the # portion can be quenched by quenching only fluorescence with a single photon laser of 488 nm wavelength, and the ★ portion was irradiated with a 780 nm wavelength diphoton laser to confirm that only the nucleic acid in the complex can be separated and photodegraded. . Therefore, C in FIG. 4 quenched fluorescence, but fluorescence can be observed when a new probe is attached, and D is irradiated with a two-photon laser having a near-infrared wavelength. By confirming that it was not, it was confirmed that the nucleic acid was separated by photolysis by responding in the near infrared.
본 발명의 실시예 1 내지 실시예 2 및 실험예 1 내지 실험예 3을 종합하면, 본 발명의 화합물을 사용하여 핵산의 합성, 분리 및 복합체 형성이 가능하고 종래대비 추가공정을 줄여 경제적인 효과를 제공하며 UV뿐만 아니라 근적외선에서도 광분해하여 세포의 손상을 줄이고 좁은 영역에도 활용될 수 있는 것을 확인할 수 있다.Synthesizing Examples 1 to 2 and Experimental Examples 1 to 3 of the present invention, it is possible to synthesize, separate, and complex a nucleic acid using the compounds of the present invention, and reduce the additional process compared to the prior art to reduce the economic effect. It can be confirmed that it can be utilized in narrow areas by reducing cell damage by providing photolysis in the near infrared as well as UV.
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.From the above description, those skilled in the art to which the present invention pertains will appreciate that the present invention may be implemented in other specific forms without changing its technical spirit or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the following claims rather than the detailed description and equivalent concepts thereof.
Claims (12)
- 하기 화학식 1로 표시되는 화합물:Compound represented by the formula (1):[화학식 1][Formula 1]
상기 화학식 1에서,In Chemical Formula 1,R1은 수소 또는 C1-6알킬,R 1 is hydrogen or C 1-6 alkyl,R2a 내지 R2b는 각각 독립적으로 C1-6알킬,R 2a to R 2b are each independently C 1-6 alkyl,n1은 0 내지 40에서 선택되는 정수,n 1 is an integer selected from 0 to 40,n2는 0 내지 40에서 선택되는 정수,n 2 is an integer selected from 0 to 40,L은L is
또는or
상기 m은 1 내지 3에서 선택되는 정수.The m is an integer selected from 1 to 3. - 제1항에 있어서,According to claim 1,상기 화합물은 광반응성 핵산 합성 또는 핵산 분리 용도로 사용되는 것인 화합물.The compound is used for photoreactive nucleic acid synthesis or nucleic acid separation.
- 제1항에 있어서,According to claim 1,상기 화합물은 하기 화학식 2, 화학식 3, 또는 화학식 4로 표시되는 것인 화합물:The compound is a compound represented by the following formula 2, formula 3, or formula:[화학식 2][Formula 2]
[화학식 3][Formula 3]
[화학식 4][Formula 4]
상기 화학식 2 내지 화학식 4 에서,In Chemical Formulas 2 to 4,R1은 수소 또는 C1-6알킬,R 1 is hydrogen or C 1-6 alkyl,R2a 내지 R2b는 각각 독립적으로 C1-6알킬,R 2a to R 2b are each independently C 1-6 alkyl,n은 0 내지 40에서 선택되는 정수,n is an integer selected from 0 to 40,m은 1 내지 3에서 선택되는 정수.m is an integer selected from 1 to 3. - 하기 화학식 1의 화합물을 포함하는 광반응성 핵산 합성용 조성물:A composition for synthesizing a photoreactive nucleic acid comprising the compound of Formula 1:[화학식 1][Formula 1]
R1은 수소 또는 C1-6알킬,R 1 is hydrogen or C 1-6 alkyl,R2a 내지 R2b는 각각 독립적으로 C1-6알킬,R 2a to R 2b are each independently C 1-6 alkyl,n1은 0 내지 40에서 선택되는 정수,n 1 is an integer selected from 0 to 40,n2는 0 내지 40에서 선택되는 정수,n 2 is an integer selected from 0 to 40,L은L is
또는or
상기 m은 1 내지 3에서 선택되는 정수.The m is an integer selected from 1 to 3. - 제4항에 있어서,According to claim 4,상기 조성물은 화학식 1의 화합물에 포함된 비닐기를 통해 고분자와 결합하는 것인, 핵산 합성용 조성물.The composition is a composition for synthesizing nucleic acids, which is to be combined with a polymer through a vinyl group included in the compound of Formula 1.
- 제4항에 있어서,According to claim 4,상기 조성물은 화학식 1의 화합물에 포함된 포스포르아미다이트 모이어티를 통해 핵산과 접합되는 것인, 핵산 합성용 조성물.The composition is to be conjugated with a nucleic acid through a phosphoramidite moiety contained in the compound of Formula 1, composition for nucleic acid synthesis.
- 제4항에 있어서,According to claim 4,상기 조성물은 적외선 조사시 화학식 1의 화합물에 포함된 쿠마린에 의해 이광자 흡수를 통해 합성된 핵산을 분리하는 것인, 핵산 합성용 조성물.The composition is to separate nucleic acids synthesized through diphoton absorption by coumarin contained in the compound of Formula 1 when irradiated with infrared rays.
- 하기 화학식 1로 표기되는 화합물과 제1핵산을 반응시켜 화학식1의 포스포르아미다이트 모이어티에 제1핵산이 결합된 제1복합체를 제조하는 제1단계; 및A first step of preparing a first complex in which a first nucleic acid is bound to a phosphoramidite moiety of Formula 1 by reacting a compound represented by Formula 1 with a first nucleic acid; And상기 제1복합체를 제2핵산과 접촉시켜 상보적인 서열의 핵산 이중가닥을 형성하는 제2복합체를 제조하는 제2단계;를 포함하는 것인, 핵산 제조방법:A second step of preparing a second complex that forms a double-stranded nucleic acid of a complementary sequence by contacting the first complex with a second nucleic acid;[화학식 1][Formula 1]
상기 화학식 1에서,In Chemical Formula 1,R1은 수소 또는 C1-6알킬,R 1 is hydrogen or C 1-6 alkyl,R2a 내지 R2b는 각각 독립적으로 C1-6알킬,R 2a to R 2b are each independently C 1-6 alkyl,n1은 0 내지 40에서 선택되는 정수,n 1 is an integer selected from 0 to 40,n2는 0 내지 40에서 선택되는 정수,n 2 is an integer selected from 0 to 40,L은L is
또는or
상기 m은 1 내지 3에서 선택되는 정수.The m is an integer selected from 1 to 3. - 제8항에 있어서,The method of claim 8,상기 제2핵산은 제1핵산의 일부 또는 전부와 상보적인 서열을 갖는 단일가닥 핵산 또는 A, G, C 및 T 핵산 단량체의 혼합물인 것인, 핵산 제조방법.The second nucleic acid is a single-stranded nucleic acid having a sequence complementary to some or all of the first nucleic acid or a mixture of A, G, C and T nucleic acid monomers, nucleic acid production method.
- 제8항에 있어서,The method of claim 8,상기 제2단계 이전 또는 이후에 화학식1의 비닐기에 고분자 네트워크겔을 결합시켜 제3복합체를 형성하는 제3단계를 추가로 포함하는 것인, 핵산 제조방법.A method of manufacturing a nucleic acid, further comprising a third step of forming a third complex by bonding a polymer network gel to a vinyl group of Formula 1 before or after the second step.
- 제10항에 있어서,The method of claim 10,상기 제3단계 이후 상기 상기 화합물에 광을 조사하여 핵산을 분리하는 단계를 더 포함하는 것인, 핵산 제조방법.After the third step further comprising the step of separating the nucleic acid by irradiating the compound with light, nucleic acid production method.
- 제11항에 있어서,The method of claim 11,상기 광은 자외선, 가시광선 또는 적외선인 것인, 핵산 제조방법. The light is ultraviolet, visible or infrared, nucleic acid production method.
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| GOGUEN, B. N. ET AL.: "Sequential Activation and Deactivation of Protein Function Using Spectrally Differentiated Caged Phosphoamino Acids", J. AM. CHEM. SOC., vol. 133, 2011, pages 11038 - 11041 * |
| SUN, H. ET AL.: "Coumarin-Induced DNA Ligation, Rearrangement to DNA Interstrand Crosslinks, and Photorelease of Coumarin Moiety", CHEMBIOCHEM ., vol. 17, 2016, pages 2046 - 2053 * |
| SUN, H. ET AL.: "Quantitative DNA Interstrand Cross-Link Formation by Coumarin and Thymine: Structure Determination, Sequence Effect, and Fluorescence Detection", J. ORG . CHEM. 2014, vol. 79, pages 11359 - 11369 * |
| WANG, F. ET AL.: "Dually Gated Polymersomes for Gene Delivery", NANO LETT., vol. 18, 27 July 2018 (2018-07-27), pages 5562 - 5568 * |
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