KR20080015338A - Photolabile compound and substrate for oligomer probe array with the same - Google Patents

Abstract

A photo-labile compound is provided to increase a photolysis reaction rate, and thus improve total reaction yield when applied to a manufacture process of oligomer probe array. A photo-labile compound has a structure represented by the following formula 1, wherein R1 is hydrogen, an alkyl group having 1-3 carbon atoms, or an alkoxy group having 1-3 carbon atoms, X is the formula (a) or (b), and Y is the following formula (Y).

Description

Photolabile compound and substrate for oligomer probe array with the same

1 to 3 are cross-sectional views of a substrate for an oligomeric probe array according to various embodiments of the present invention coupled with a photodegradable compound directly or via a linker to the substrate according to one embodiment of the invention.

(Explanation of symbols for the main parts of the drawing)

100: oligomer probe array 110: substrate

120: probe cell active 130: probe cell separation region

140: linker 150: functional group

152: photodegradable protecting group of photodegradable compound 165: oligomer probe

The present invention relates to a photodegradable compound and a substrate for an oligomeric probe array combined with the photodegradable compound.

The oligomeric probe array refers to a high density arraying of oligomer probes on a solid support such as a silicon wafer substrate.

The oligomeric probe array is, for example, exposed to a functional group by degassing a protecting group of a specific region through light irradiation on a substrate on which a functional group protected by photolabile protecting groups is fixed, and then, oligonucleotide and polypeptide, Oligomeric probes such as peptide nucleic acid (PNA) may be prepared by a method of binding to a substrate. The method of binding the oligomer probe to the substrate may include photolithography, which is a method of synthesizing monomers in-situ, or a spotting method of binding a previously synthesized oligomer probe to a substrate.

Photodegradable protecting groups protect reactive functional groups until they are deprotected by exposure to constant light. The rate of deprotection of photodegradable protecting groups can affect the overall yield.

Photodegradable protecting groups may be used in the preparation of the oligomeric probe array, and the photolysis reaction rate may vary depending on the type of the photodegradable protecting groups. Efforts have been made to develop photodegradable compounds that can provide photodegradable protecting groups to increase the overall reaction yield.

The technical problem to be achieved by the present invention is to provide a new photodegradable compound that can increase the photolysis reaction rate.

In addition, another technical problem to be achieved by the present invention is to provide a substrate for an oligomeric probe array to which a photodegradable compound is bound.

Technical problems of the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned will be clearly understood by those skilled in the art from the following description.

Photodegradable compound according to an embodiment of the present invention for achieving the above technical problem is represented by the following formula.

(Wherein R ₁ is hydrogen, an alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms,

, 또는

이되, X is

, or

This,

R ₂ , R ₃ , R ₄ , R _{5 and} R ₆ are each hydrogen, an alkyl group having 1 to 3 carbon atoms or an alkoxy group having 1 to 3 carbon atoms,

,

Y is

,

,

또는

이고,

,

or

ego,

B is adenine, cytosine, guanine, thymine, uracil,

R ₇ is hydrogen, an amino group, an alkyl group or a phosphine

R ₈ is hydrogen, a hydroxyl group, -OR ₁₀ or SR ₁₀ , wherein R ₁₀ is an alkyl, alkenyl, acetal or silyl ether group,

R ₉ is an alkyl group, a phenyl group, or sulfur,

P is 0 to 5,

Q is 0 to 10.)

The oligomer probe array substrate according to an embodiment of the present invention for achieving the above another technical problem is a substance of the following formula is bonded directly or via a linker to the substrate.

(Wherein R ₁ is hydrogen, an alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms,

, 또는

이되,X is

, or

This,

R ₂ , R ₃ , R ₄ , R _{5 and} R ₆ are each hydrogen, an alkyl group having 1 to 3 carbon atoms or an alkoxy group having 1 to 3 carbon atoms,

Y is halogen, hydrogen

이고, or

ego,

Q is 0 to 10.)

Specific details of other embodiments are included in the detailed description and the drawings.

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various forms, and only the present embodiments are intended to complete the disclosure of the present invention, and the general knowledge in the art to which the present invention pertains. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention is defined only by the scope of the claims.

Thus, in some embodiments, well known process steps, well known structures and well known techniques are not described in detail in order to avoid obscuring the present invention.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In this specification, the singular also includes the plural unless specifically stated otherwise in the phrase. As used herein, including and / or containing includes the presence or addition of one or more other components, steps, operations and / or elements other than the components, steps, operations and / or elements mentioned. Use in the sense that does not exclude. And ″ and / or ″ include each and all combinations of one or more of the items mentioned. In addition, like reference numerals refer to like elements throughout the following specification.

Hereinafter, a photodegradable compound according to embodiments of the present invention will be described.

Photodegradable compounds according to embodiments of the present invention are represented by the following formula.

(Wherein R ₁ is hydrogen, an alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms,

, 또는

이되, X is

, or

This,

R ₂ , R ₃ , R ₄ , R _{5 , and} R ₆ each represent hydrogen, an alkyl group having 1 to 3 carbon atoms, or an alkoxy group having 1 to 3 carbon atoms.)

Portions other than Y in the above formula may be referred to as photodegradable protecting groups. A photolabile protective group refers to a group that is attached to a reactive functional group so that no chemical reaction occurs until it is removed through irradiation of UV or visible light. When a photodegradable protecting group is removed and a reactive functional group is exposed, a chemical reaction occurs, which may be referred to as deprotection. A compound including a photodegradable protecting group may be referred to as a photolabile compound.

In some embodiments of the invention, Y in the formula may be a halogen, or hydroxyl group. The photodegradable compound when Y is a halogen or hydroxyl group may then be combined with nucleosides, peptide nucleic acids, linkers and the like to constitute monomers or linkers used in the preparation of oligomer probes.

In some other embodiments of the present invention, Y may be a nucleoside or a nucleotide.

또는

일 수 있다.More specifically, Y

or

Can be.

Wherein B is adenine, cytosine, guanine, thymine or uracil,

R ₇ is hydrogen, an amino group, an alkyl group or a phosphine,

R ₈ is hydrogen, a hydroxyl group, -OR ₁₀ or SR ₁₀ , and R ₁₀ is an alkyl, alkenyl, acetal or silyl ether group.)

More preferably, R ₇ may be a photodegradable compound which is hydrogen or a phosphite amide group represented by the following formula. The OH group of the 3 'moiety or the 5' of the ribose sugar and the deoxyribose sugar may be free or protected with a protecting group and R ₇ may be a phosphite amide of the formula

or

(Wherein R ₁₁ has the same or different independent linear or branched alkyl radicals of 1 to 4 carbon atoms.)

R ₄ of this photodegradable compound is an O-methyl radical, an O-ethyl radical, an O-aryl radical, an O-tetrahydropyranyl, an O-methoxytetrahydropyranyl ralical, an Ot-butyldimethylsilyl radical. Can be.

Nucleosides and nucleotides include known purine and pyrimidine bases, as well as methylated purines or pyrimidines, acylated purines or pyrimidines, and the like. In addition, nucleosides and nucleotides include not only conventional ribofuranose and deoxyrifofuranose, but also modified sugars in which one or more hydroxyl groups are substituted with halogen atoms or aliphatic groups, or functional groups such as ethers. It may include. Photodegradable protecting groups may be bonded to such nucleosides and nucleotides, and derivatives thereof to form photodegradable compounds.

In some other embodiments of the invention Y is

,

또는

일 수 있다.

,

or

Can be.

Provided that B is adenine, cytosine, guanine, thymine, uracil,

R ₉ is an alkyl group, a phenyl group, or sulfur,

P is 0 to 5, and q is 0 to 10).

Preferably q may be 3 to 10.

The photodegradable compound may be a linker molecule interposed between the oligomer probe and the substrate. The linker may provide the spatial margin required for hybridization of the oligomeric probe with the sample in the oligomeric probe array. That is, it can be used to provide an optimal length between the synthesized oligomer probe and the substrate.

Hereinafter, the oligomeric probe array substrate to which the above-mentioned photodegradable compound is bonded is demonstrated.

1 to 3 are cross-sectional views of a substrate for an oligomeric probe array according to various embodiments of the present invention coupled with a photodegradable compound directly or via a linker to a substrate in accordance with some embodiments of the present invention.

Oligomeric probe array substrate (100, 101, 102) according to embodiments of the present invention is a material of the following formula is bonded directly or via a linker to the substrate.

(Wherein R ₁ is hydrogen, an alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms,

, 또는

이되,X is

, or

This,

R ₂ , R ₃ , R ₄ , R _{5 and} R ₆ are each hydrogen, an alkyl group having 1 to 3 carbon atoms or an alkoxy group having 1 to 3 carbon atoms,

Y is halogen, hydrogen

이고, or

ego,

Q is 0 to 10.)

The substrate for the oligomeric probe array refers to a substrate for the preparation of the oligomeric probe array. The oligomeric probe array refers to a high density arraying of oligomer probes on a solid support such as a silicon wafer substrate. The oligomeric probe refers to a polymer having a molecular weight of approximately 1,000 or less in a polymer composed of two or more monomers covalently bonded, but is not limited thereto. The oligomeric probe may comprise about 2-500 monomers, preferably 5-30 monomers. The monomer may be a nucleoside, nucleotide, amino acid, peptide or the like depending on the type of probe. The oligomeric probe 165 may be synthesized in advance or may be synthesized through an in-situ photolithography process on the active phase.

The substrate 110 may be made of a material capable of minimizing unwanted non-specific binding and substantially zero during hybridization. In addition, the substrate 110 may be made of a material that is transparent to visible light and / or UV. The substrate may be a flexible or rigid substrate. The flexible substrate may be a membrane or plastic film such as nylon, nitrocellulose, or the like. The rigid substrate may be a silicon substrate, a transparent glass substrate such as soda lime glass, or the like. In the case of a silicon substrate or a transparent glass substrate, there is an advantage that little non-specific binding occurs during the hybridization process. Moreover, in the case of a transparent glass substrate, it is transparent to visible light and / or UV, and is advantageous for the detection of a fluorescent substance. The silicon substrate or the transparent glass substrate has an advantage that the manufacturing process and the photolithography process of various thin films which are already stably established and applied in the manufacturing process of the semiconductor device or the manufacturing process of the LCD panel can be applied as they are.

1 to 3, the oligomer probe array substrate 100, 101, 102 may be a substrate to which the photodegradable protecting group 152 of the photodegradable compound described above is coupled through a linker molecule (linker molecule). Can be. The linker 140 may provide the spatial margin necessary for hybridization of the oligomer probe and the sample in the oligomeric probe array. That is, it can be used to provide an optimal length between the synthesized oligomer probe and the substrate. The linker 140 may be a silane linker or nanoparticles.

As shown in FIG. 1, a substrate according to an exemplary embodiment of the present invention has a probe cell active 120 formed on a surface of the substrate 110 and a linker 140 interposed between the probe cell active 120. And a photodegradable protecting group 152 of a photodegradable compound may be coupled to the functional group 150 of the linker 140. The region surrounding the probe cell active region 120 and to which the linker 140 is not coupled is called the probe cell isolation region 130.

The probe cell active 120 is preferably formed of a material that is substantially stable without hydrolysis upon contact with hybridization assay conditions, such as pH 6-9 phosphate or TRIS buffer. Accordingly, the probe cell active 120 may be a silicon oxide film such as a spin-on siloxane film, a PE-TEOS film, an HDP oxide film or a P-SiH4 oxide film, a thermal oxide film, silicate such as hafnium silicate, zirconium silicate, silicon nitride film, or silicon oxynitride film. , Metal oxynitride films such as hafnium oxynitride films and zirconium oxynitride films, titanium oxide films, tantalum oxide films, aluminum oxide films, metal oxide films such as hafnium oxide films, zirconium oxide films and ITO, metals such as polyimide, polyamine, gold, silver, copper, and palladium Or polymers such as polystyrene, polyacrylic acid, polyvinyl, and the like. Preferably, the probe cell active 120 may be formed of a material that is stably applied in a semiconductor manufacturing process or an LCD manufacturing process.

2, the immobilization layer 121 is formed on the surface of the substrate 110, and the linker 141 is interposed on the immobilization layer 121. The photodegradable protecting group 152 of the photodegradable compound may be coupled to the functional group 150 of 141.

3, the probe cell active 120 is formed on a surface of the substrate 110, and the nanoparticles 130 are interposed on the probe cell active 120. After the linker 140 is interposed between the nanoparticles 130 and the photodegradable protecting group 152 of the photodegradable compound is coupled to the functional group 150 of the linker.

If the photodegradable compound including the photodegradable protecting group 152 has the function of the linker, the formation of the linker 140 is omitted, and the photodegradable compound is formed in the probe cell active 120, the immobilization layer 121, or the nanoparticles ( 130) directly.

Since the photodegradable protecting group 152 of the photodegradable compound in the oligomer probe array substrates 100, 101, and 102 according to the embodiments of the present invention has a fast deprotection reaction rate, that is, a half-life during irradiation Is short. Therefore, when using the oligomer probe array substrate (100, 101, 102) according to the embodiments of the present invention for the production of the oligomer probe array, it is possible to increase the overall reaction yield.

Hereinafter, a method for preparing a photodegradable compound according to embodiments of the present invention will be described.

<Scheme 1>

First, according to Scheme 1, a compound having a halogenitrophenyl group (halonitrophenyl) can be synthesized.

Reaction of starting compound 3-aminoacetophenone (3'-aminoacetophenone) with methyl trifluoroacetate in methanol solvent to synthesize protected compound # 2 followed by nitrification using sulfuric acid and nitric acid React After deprotection with ammonia water to obtain compound 4, compound 5 is obtained through a Sandmeyer reaction. The reaction is continued with sodium borohydride to obtain compound 6. Each step can be performed using silica gel column chromatography.

Thereafter, palladium coupling derivatives may be formed according to Scheme 2 and Scheme 3.

<Scheme 2>

<Scheme 3>

Palladium catalyst (palladium catalyst) can be used to synthesize a derivative with a variety of functional groups. In particular, various derivatives can be synthesized using acetylene derivatives, and furthermore, unlocalized derivatives can be synthesized using continuous palladium catalysts.

Next, the alcohol formed in the above process may be reacted with a phosgene and a phosgene derivative as in Scheme 4 and Scheme 5 to obtain a photodegradable compound including oxycarbonyl chloride.

<Scheme 4>

Scheme 5

Preferably it is reacted at -20 ℃ to +25 ℃ in a non-polar organic solvent. In addition to phosgene, diphosgene (chloroformic acid trichloromethyl ester) or triphosgene (bistrichloromethyl carbonate) may be used.

On the other hand, the case in which Y is nucleoside in the following formula can be prepared by the method shown in Scheme 6 and 7.

(Wherein R ₁ is hydrogen, an alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms,

, 또는

이되, X is

, or

This,

R <_2> , R <_3> , R <_4> , R <_{5> and} R <_6> are hydrogen, a C1-C3 alkyl group, or a C1-C3 alkoxy group, respectively.

,

Y is

,

B is adenine, cytosine, guanine, thymine, uracil,

R ₇ is hydrogen, an amino group, an alkyl group or a phosphine,

R ₈ is hydrogen, a hydroxyl group, -OR ₁₀ or SR ₁₀ , and R ₁₀ is an alkyl, alkenyl, acetal or silyl ether group.)

<Scheme 6>

Scheme 7

After synthesizing the photodegradable protecting group represented by the above formula, the excess phosgene derivative and the solvent are first distilled under vacuum, and then the photodegradable protecting group having a chlorocarboxylic acid ester group is reacted with the nucleoside.

A solvent mixture consisting of dichloromethane and a polar organic solvent may be reacted with the nucleoside and chlorocarboxylic acid ester of the photodegradable protecting group at −60 ° C. to + 25 ° C. with any base selected. DMF or pyridine is preferred as a polar organic solvent and when pyridine is used no addition of other base is necessary. However, if dichloromethane / DMF solvent mixtures are used, pyridine bases such as pyridine, trietyl amine or ethyl diisopropyl amine are preferred. Although the mixing ratio of dichloromethane to pyridine or to DMF can not be precisely stated, it is preferable that the volume of dichloromethane is 1 or 3 times the volume of each of pyridine or DMF respectively.

The nucleosides are dissolved in pyridine or DMF / salts and dropped in chlorocarboxylic acid ester solution and the appropriate reaction temperature is adjusted. The molar ratio of nucleic acid to chlorocarboxylic acid ester can be matched one-to-one by stoichiometry. Preferably, the molar ratio of nucleoside to chlorocarboxylic acid ester can be adjusted one-to-one or one-to-one by increasing the amount of chlorocarboxylic acid ester.

When the reaction is completed after about 5 to 6 hours, the nucleosides of the present invention can be separated and purified by known methods. For example, after dilution with dichloromethane, the salt is washed to remove the salt, dried by an organic phase, and then concentrated or crystallized in accordance with a known method such as silica gel chromatography. Can be isolated or purified. In this way it is possible to obtain high purity nucleoside derivatives with a yield of about 70-80%.

According to a preferred embodiment of the present invention, the phosphite amide group represented by the following formula is selected from the 3 ′ position (R ₁₁ ) of the nucleoside derivative according to a known method. Position).

, 또는

, or

Provided that R ₁₁ has the same or different independent linear or branched alkyl radicals of 1 to 4 carbon atoms.

Generally, this reaction is reacted at 0 ° C. to 25 ° C. in a solvent mixture comprising dichloromethane and acetonitrile using an appropriate phosphine as the catalyst and 1 H tetrazol. Preferably the phosphine is used in an excess molar concentration of 1.5 to 3 times. The molar ratio of phosphine to 1H tetrazole can be adjusted from 2 to 4 times. The quantitative ratio of dichloromethane to acetonitrile is not relatively critical and preferably 1 to 1 to 4 to 1 is preferred.

More detailed information about the present invention will be described through the following specific experimental examples, and details not described herein will be omitted because it can be inferred technically by those skilled in the art.

Experimental Example  One - Halogenoniphenyl  Compound synthesis halonitrophenyl compound )

a) 3-acetyl- Trifluoroacetylaminobenzene  (3- Acetyl -trifluoroacetylaminobenzene) Synthesis

In a 100 mL round bottom flask, 10 mmol of the starting material, 3-aminoacetophene (3'-aminoacetophenone), was dissolved in methanol, and then added dropwise at room temperature for 30 minutes with methyl trifluoroacetate (1.2eq), followed by 2 Stir for hours. After confirming by TLC that the reaction was completed, a small amount of water was used to terminate the reaction. The solvent was then removed under reduced pressure, and the residue was dissolved in ethyl acetate (50 mL), washed with water (30 mL, twice), dried over anhydrous sodium sulfate and distilled under reduced pressure to obtain the desired 3-acetyl-trifluoroacetylamino. Benzene (85%) was obtained as a brown oil.

Rf = 0.39 (Hexane / EtOAc = 1/1), 1 H NMR (300 MHz, CDCl 3) δ 7.70 (s, 1H, NH), 7.33-7.14 (m, 4H, aromatic), 2.13 (s, 3H)

b) 3-acetyl-4-nitro- Aminobenzene (3- Acetyl -4- nitro - aminobenzene ) synthesis

Starting material (3-acetyl-trifluoroacetylaminobenzene, 10 mmol) was added slowly to conc H 2 SO 4 (20 mL) at −20 ° C., stirred for 30 minutes, and then nitric acid was added dropwise for 10 minutes. The reaction was continuously stirred for 2 hours and poured into ice water when the reaction was complete. Ice water was extracted with ethyl acetate (50 mL, 2 times), the organic layer was washed with sodium hydrogen carbonate solution, the organic layer was dried over anhydrous sodium sulfate, distilled under reduced pressure, ethyl acetate was removed, impure, and then placed in conc NH4OH. After stirring for 1 hour, the reaction was evaporated using a rotary, extracted with EtOAc and water, and then purified with silica gel column chromatography (Hexane / EtOAc = 3/1) to obtain the desired 3-acetyl-4-nitro-aminobenzene. To a brown solid (54%).

Rf = 0.25 (Hexane / EtOAc = 3/1), 1 H NMR (300 MHz, CDCl 3) δ 7.95 (m, 1 H), 6.44 (m, 2H), 3.99 (br, 2H, NH 2), 2.11 (s, 3H)

c) 3-acetyl-4-nitro- Bromobenzene  (3- Acetyl -4- nitro - bromobenzene  )synthesis

In an ice bath (3-acetyl-4-nitro-aminobenzene, 10 mmol) was added dropwise to HBr / H ₂ O (100 mL, 1/2, v / v) and sodium nitrite solution was slowly added dropwise. . The reaction was stirred at 60 ° C. for 2 hours, and when the reaction was completed, the precipitate was filtered, the filtrate was neutralized with 1N NaOH, extracted with EtOAc, dried over anhydrous sodium sulfate, distilled under reduced pressure and silica gel column chromatography (Hexane / EtOAc = 2). / 1) to afford the desired 3-acetyl-4-nitro-bromobenzene as a yellow oil (75%).

Rf = 0.31 (Hexane / EtOAc = 2/1), 1 H NMR (300 MHz, CDCl3) δ 7.74 (d, 1H), 7.52 (d, 1H), 7.44 (dd, 1H), 2.11 (s, 3H)

d) 1- (5- Bromo -2- Nitrophenyl Ethanol (1- (5- Bromo -2- nitrophenyl ) ethanol  6) synthetic

In an ice bath, 3-acetyl-4-nitro-bromobenzene (10 mmol) was added to a 100 mL two neck round flask, dissolved in a solvent ether (30 mL), and sodium borohydride was slowly added dropwise. The reaction was stirred at room temperature for 2 hours, the reaction was confirmed by TLC, the reaction was terminated with water (5 mL), the precipitate was filtered off, the filtrate was washed with sodium chloride solution (30 mL, 2 times), dried over anhydrous sodium sulfate, and then distilled under reduced pressure. The desired 1- (5-bromo-2-nitrophenyl) ethanol was obtained as a yellow oil (71%) using silica gel column chromatography (Hexane / EtOAc = 2/1).

Rf = 0.33 (Hexane / EtOAc = 1/3), 1 H NMR (300 MHz, CDCl 3) δ 7.73 (d, 1H), 7.52 (d, 1H), 7.42 (dd, 1H), 6.15 (m, 1H), 1.79 (br, 1H), 1.62 (d, 3H)

Experiment 2 - palladium couplings (Palladium coupling ) derivatives synthesis

a) (E) -1- (5-1,2- Diphenylvinyl )-2- Nitrophenyl ) Ethanol ((E) -1- (5- (1,2- diphenylvinyl ) -2-nitrophenyl) ethanol) Synthesis

Nitrobromophenyl (nitrobromophenyl, 1mmol) was put in a pressure tube, dissolved in a solvent (DMF, 20mL), and 5% Pd (OAc) 2, 1eq LiCl, 1.2eq KOAc was slowly added to the reaction solution, followed by acetylene ( acetylene (1.5 eq) derivatives are added and sealed. The reaction proceeded for 3 hours at 80 ℃ and the completion of the reaction was confirmed using TLC. At the end of the reaction, the reaction was quenched with water. The reaction was extracted with NH 4 Cl (aq, 50 mL) and EtOAc (50 mL). The desired compound was decanted into an organic layer, washed once with sodium chloride solution (50 mL), and dried. After drying over sodium sulfate, the solvent was distilled off under reduced pressure to obtain the target compound. The resulting silica gel column chromatography (Hex / EtOAc = 1/4) was used to (E) -1- (5-1,2-diphenylvinyl)- 2-nitrophenyl) ethanol was obtained.

 Rf = 0.32 (Hexane / EtOAc = 1/2), IR (neat) 1599, 1622 Cm-1, 1H NMR (300MHz, CDCl3) δ 7.73 (d, 1H), 7.53 (m, 3H), 7.46-7.22 ( m, 9H), 6.81 (s, 1H), 6.14 (m, 1H), 1.80 (br, 1H), 1.62 (d, 3H)

b) 1- (2-nitro-5- (10- Phenylphenanthrene -9-day) Phenyl ) Ethanol (1- (2- nitro -5- (10- phenylphenanthren -9- yl ) phenyl ) ethanol ) synthesis

2-nitro-5- (2-phenylethynyl) phenyl) ethanol ((2-nitro-5- (2-phenylethynyl) phenyl) ethanol, 1 mmol) was placed in a pressure tube and iodibiphenyl, 1.1eq), add 1eq LiCl and 2eq NaOAc, dissolve in solvent DMF (20mL), seal, and then stir the reaction at 100 ° C for 30 hours. After confirming the termination of the reaction by TLC, when the end of the reaction, ammonium chloride (aq, sat, 50 mL) and EtOAc (50 mL) were used to lubricate the desired compound to the organic layer, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. And 1- (2-nitro-5- (10-phenylphenanthrene-9-yl) phenyl) ethanol (1- (2-nitro) using final silica gel column chromatography (Hex / EtOAc = 1/1) -5- (10-phenylphenanthren-9-yl) phenyl) ethanol) was obtained.

Rf = 0.35 (Hexane / EtOAc = 1/1)

Experimental Example  3- Oxycarbonyl  Chloride ( oxycarbonyl chloride ) synthesis

The reactant (1eq) and triethylamine (1eq) were dissolved in the dried THF solution, and then trichloromethyl chloroformate (1.2eq) was added dropwise at 0 ° C. for 5 minutes while dissolved in THF. After the reaction was stirred at 0 ° C. for 1 hour, the resulting precipitate was filtered and the filtrate was fractionally distilled to obtain a desired compound (nitrophenylchloroformate derivative).

Experimental Example 4-of formula Photodegradable  Having protecting group Nucleoside  Produce

A photodegradable protecting group and a nucleoside bound to a base were reacted. First, the base-bound nucleoside (10 mmol) was distilled under reduced pressure using a rotary evaporator three times using dried pyridine. Then, it was dissolved in pyridine (20 mL), and nitrophenylchloroformate derivative (10.2 mmol) was slowly added dropwise under N 2 (g) at -10 ° C. DMAP (0.1eq, in 5mL pyridine) was added continuously and stirred for 5 hours using a stirring bar. The reaction was confirmed by TLC, and when the reaction was completed, the pyridine was removed under reduced pressure, dissolved in EtOAc (50 mL), washed once with NaHCO 3 (aq, sat, 50 mL), washed once with NaCl (aq, sat, 30 mL), and the organic layer was washed. After drying over Na 2 SO 4 and distillation under reduced pressure, a desired compound (Photolabile protective deoxynucleoside) was obtained using silica gel column chromatography.

Research( irradiation )Experiment

1. Run

Photolabile protective deoxynucleoside (dT) derivatives coupled to photodegradable protecting groups were dissolved in acetonitrile (acetonitrile / H2O = 95/5 (v / v) at a concentration of 100 uM and then 365 nm at 200 W using a Hg (Xe) ARC lamp. 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, and 1 minute of exposure were performed to investigate the degree of deprotection using HPLC. According to HPLC (Varian MicroPak SP column (C18)) was analyzed using a solvent of ACN: H 2 O = 1: 1.5 and the flow rate (flow rate) was 1 mL / min.

Comparative Example 1 is a nucleic acid (deoxythymidine, dT) having methyl-6-nitropiperonyloxycarbonyl (MeNPOC, methyl-6-nitroperonyloxycarbonyl) as a photodegradable protecting group.

Comparative Example 2 is a nucleic acid (deoxythymidine, dT) having 3'-nitrophenylpropyloxycarbonyl (NPPOC, 3'-Nitrophenylpropyloxycarbonyl) as a photodegradable protecting group.

Compound 1 to compound 2 are compounds represented by the following formula.

<Compound 1>

<Compound 2>

(B is thymidine.)

Table 1 is a table showing the results of the investigation experiment numerically, shows the HPLC analysis results according to the exposure time.

Table 1 Results of Investigation Experiment

compound Solvent (v / v) t _1/2 (second) One Comparative Example 1 (MeNPOC-dT) ACN / H ₂ O = 95/5 37 2 Comparative Example 2 (NPPOC-dT) ACN / H ₂ O = 95/5 33 3 Compound 1 (1- (5-((E) -1,2-diphenylvinyl) -2-nitrophenyl) ethyl ((2R, 3R, 5R) -3-hydroxy-5- (5-methyl-2,4-dioxo -3,4-dihydropyrimidin-1 (2H) -yl) -tetrahydrofuran-2-yl) methyl carbonate) ACN / H ₂ O = 95/5 31 4 Compound 2 ((((2R, 3R, 5R) -3-hydroxy-5- (5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1 (2H) -yl) -tetrahydrofuran-2-yl) methyl 1- (2-nitro-5- (10-phenylphenanthren-9-yl) phenyl) ethyl carbonate) ACN / H ₂ O = 95/5 27

Irradiation experiments were performed to analyze HPLC over exposure time. Compounds synthesized in the present invention showed a faster rate of degradation than conventional NPPOC-, MeNPOC- photodegradable protecting groups widely used.

Although the embodiments of the present invention have been described above, it will be understood by those skilled in the art that the present invention may be implemented in other specific forms without changing the technical spirit or essential features thereof. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

As described above, the photodegradable compound according to the present invention may be used for preparing an oligomeric probe array. Since the substrate for the oligomeric probe array to which the compound represented by the formula or the material represented by the formula is bound is fast to deprotect the included photodegradable protecting group, the overall reaction yield may be increased when used in the oligomeric probe array manufacturing process.

Claims (8)

Photodegradable compound represented by the following formula.

(Wherein R ₁ is hydrogen, an alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms, X is

, or

This, R ₂ , R ₃ , R ₄ , R _{5 and} R ₆ are each hydrogen, an alkyl group having 1 to 3 carbon atoms or an alkoxy group having 1 to 3 carbon atoms, Y is

,

,

or

ego, B is adenine, cytosine, guanine, thymine, uracil, R ₇ is hydrogen, an amino group, an alkyl group or a phosphine R ₈ is hydrogen, a hydroxyl group, -OR ₁₀ or SR ₁₀ , wherein R ₁₀ is an alkyl, alkenyl, acetal or silyl ether group, R ₉ is an alkyl group, a phenyl group, or sulfur, P is 0 to 5, Q is 0 to 10.) According to claim 1, Y is

or

Phosphorus photodegradable compounds. Provided that B is adenine, cytosine, guanine, thymine, uracil, R ₇ is hydrogen, an amino group, an alkyl group or a phosphine R ₈ is hydrogen, a hydroxyl group, -OR ₁₀ or SR ₁₀ , and R ₁₀ is an alkyl, alkenyl, acetal or silyl ether group.) The method of claim 2, R ₇ is a photodegradable compound which is hydrogen or a phosphite amide group represented by the following formula.

or

(Wherein R ₁₁ has the same or different independent linear or branched alkyl radicals of 1 to 4 carbon atoms.) The method of claim 2, R ₈ is an O-methyl radical, O-ethyl radical, O-aryl radical, O-tetrahydropyranyl, O-methoxytetrahydropyranyl ralical, Ot-butyldimethylsilyl radical. According to claim 1, Y is

ego, Q is 3 to 10 photodegradable compound. A substrate for an oligomeric probe array in which a substance of the following formula is bonded directly to a substrate or through a linker.

(Wherein R ₁ is hydrogen, an alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms, X is

, or

This, R ₂ , R ₃ , R ₄ , R _{5 and} R ₆ are each hydrogen, an alkyl group having 1 to 3 carbon atoms or an alkoxy group having 1 to 3 carbon atoms, Y is halogen, hydrogen or

ego, Q is 0 to 10.) The method of claim 6, The substrate is an oligomeric probe array substrate of nylon, nitrocellulose, plastic film, silicon substrate and / or soda lime glass. The method of claim 6, The linker is a silane linker or a substrate for an oligomeric probe array nanoparticles.

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