KR20230108074A - Photo-affinity tag compound using alipatic diazirine molecule and method for synthesizing the same - Google Patents

Abstract

The present invention relates to a novel diazirine-based compound, a preparation method thereof, a composition for photoaffinity labeling using the same, and a method for photolabeling proteins. The newly synthesized diazirine-based compound in the present invention has a short and simple molecular structure, Since it is possible to induce efficient structural changes and has ease and stability when combined with a specific molecule or protein, it has a useful effect as a photoaffinity label.

Description

Photo-affinity tag compound using alipatic diazirine molecule and method for synthesizing the same}

The present invention relates to a diazirine-based compound for photo-affinity tag, a method for preparing the same, a composition for photo-affinity labeling using the same, and a method for photolabeling proteins.

Photo-affinity tag is a technology that separates a specific photoreactive part from a complex by exposure to light, and then induces binding to a protein through covalent bonding. Complexes capable of photo-affinity tagging can visualize the target material in vitro, and when combined with proteins after separation of a specific site of the complex due to light exposure, a stronger bond is achieved than other bonding technologies through covalent bonding. Using this technique, it is also easily used to confirm protein-protein interactions or conjugation between other complexes.

All of the compounds synthesized as fluorescent probes through photo-crosslinking have a photoreactive group and may be involved in photo-affinity labeling. When photoreactive groups of photocrosslinking are classified by type, benzophenone, aryl azide, and diazirine are mainly used.

Most of the conventional technologies use azide or benzophenone as a photo-affinity tag, but they are highly hazardous to cells, and there is a problem that many experimental procedures are required to confirm the interaction between proteins other than the photo-affinity tag. did On the other hand, among the Azide, Benzophenone, and Diazirine molecules used in the photoaffinity labeling technology, the Diazirine molecule has low toxicity when used and has the advantage of being synthesizable as a complex with a relatively short structure. However, in the case of the previously used photo-affinity tag complex using diazirine, there is a problem in that the size of the molecule is relatively large.

Accordingly, the inventors of the present invention studied to synthesize a smaller-sized complex to solve the above problems, and as a result, a novel photoaffinity labeling complex using a diazirine molecule was prepared, and the synthesized complex was a conventional photo- Compared to Azide and Benzophenone used for affinity tag, it has a smaller molecular structure and reacts to a 355nm light source, so the risk to cells is low, and the structural stability, action efficiency and structural change efficiency are confirmed to be effective. completed the present invention.

Korean Patent Publication No. 10-2017-0058377

An object of the present invention is to provide a novel diazirine-based compound represented by Formula 1 having usefulness as a photoaffinity label.

Another object of the present invention is to provide a method for preparing the compound represented by Formula 1 above.

Another object of the present invention is to provide a composition for photo-affinity tag using the compound represented by Formula 1 and a method for photo-labeling proteins.

In order to achieve the above purpose,

The present invention provides a diazirine-based compound represented by Formula 1 below or a pharmaceutically acceptable salt thereof:

[Formula 1]

(In Formula 1,

R ¹ is halogen, azido or C ₁ -C ₅ alkyloxycarbonyl;

R ² is C ₁ -C ₅ hydroxyalkyl, carboxyl, C ₁ -C ₅ alkyloxycarbonyl, or C ₂ -C ₅ alkynyl;

wherein n is an integer from 1 to 10;

Wherein m is an integer from 0 to 9).

In addition, the present invention is a method for preparing the diazirine-based compound, as shown in Scheme 1 below,

The compound represented by Chemical Formula 7 was reacted by mixing an ammonia solution, and reacted by adding hydroxylamine-O-sulfonicacid and an organic solvent, followed by triethylamine (TEA) and Adding iodine (I ₂ ) and reacting in an organic solvent to obtain a compound represented by Formula 8 (Step 1);

A compound represented by Formula 8 is mixed with imidazole, triphenylphosphine and an organic solvent, and iodine (I ₂ ) is added to react to obtain a compound represented by Formula 2 (Step 2). );

obtaining a compound represented by Chemical Formula 3 by mixing and reacting the compound represented by Chemical Formula 2 with sodium azide and an organic solvent (Step 3);

Obtaining a compound represented by Chemical Formula 4 by reacting a compound represented by Chemical Formula 3 with an organic solvent and a Jones reagent (Step 4); and

Adding magnesium chloride (MgCl ₂ ) and diethylpyrocarbonate to the compound represented by Formula 4 and reacting in an organic solvent to obtain a compound represented by Formula 5 (Step 5);

The diazirine-based compound represented by Chemical Formula 1 is an intermediate compound obtained in Steps 2 to 4 or a final compound obtained in Step 5, and a method for preparing a diazirine-based compound represented by Chemical Formula 1 is provided:

[Scheme 1]

(In Scheme 1 above,

wherein n is an integer from 1 to 10;

Wherein m is an integer from 0 to 9).

Furthermore, the present invention is a method for preparing the diazirine-based compound, as shown in Scheme 2 below,

As shown in Scheme 2 below,

The compound represented by Chemical Formula 9 is reacted by mixing an ammonia solution, reacted by adding hydroxylamine-O-sulfonicacid and an organic solvent, and then triethylamine (TEA) and Adding iodine (I ₂ ) and reacting in an organic solvent to obtain a compound represented by Formula 10 (Step 1a);

Obtaining a compound represented by Chemical Formula 11 by reacting a compound represented by Chemical Formula 10 with an organic solvent and a Jones reagent (step 4a); and

Adding magnesium chloride (MgCl ₂ ) and diethylpyrocarbonate to the compound represented by Formula 11 and reacting in an organic solvent to obtain a compound represented by Formula 6 (step 5a);

The diazirine-based compound represented by Chemical Formula 1 is the final compound obtained in step 5a, and a method for preparing the diazirine-based compound represented by Chemical Formula 1 is provided:

[Scheme 2]

(In Scheme 2 above,

wherein n is an integer from 1 to 10;

Wherein m is an integer from 0 to 9).

Furthermore, the present invention provides a photo-affinity tag composition comprising the diazirine-based compound represented by Compound 1 or a pharmaceutically acceptable salt thereof.

In addition, the present invention comprises the steps of treating a protein-containing sample with a diazirine-based compound represented by Compound 1 or a pharmaceutically acceptable salt thereof; And 300 to 380nm wavelength irradiation of light including ultraviolet (UV); provides a method for photo-labeling proteins, including.

The newly synthesized complex in the present invention binds to a protein, enabling specific binding with a specific protein and binding between a specific protein and a ligand. The complex of the present invention has a relatively simple structure and a short molecular structure compared to conventional complexes, so the efficiency of action is higher, and it is advantageous when combined with a specific molecule and has the effect of having structural stability. , and furthermore, efficient structural changes can be induced, and there is an effect that it is easy to use for the synthesis of new structures. In addition, since the light exposure wavelength of the complex used in the present invention is 355 nm, the risk to the protein structure is relatively low and the risk to cells is low.

Figure 1 shows the spectrum showing the result of proton analysis using 1H NMR (400 MHz, Chlorform-d) spectroscopy of the compound Q1 of the present invention and the chemical structure confirmed through this.
Figure 2 shows the spectrum showing the result of proton analysis using 1H NMR (400 MHz, Chlorform-d) spectroscopy of the compound Q2 of the present invention and the chemical structure confirmed through this.
Figure 3 shows the spectrum showing the result of proton analysis using 1H NMR (400 MHz, Chlorform-d) spectroscopy of the compound Q3 of the present invention and the chemical structure confirmed through this.
Figure 4 shows the spectrum showing the result of proton analysis using 1H NMR (400 MHz, Chlorform-d) spectroscopy of the compound Q4 of the present invention and the chemical structure confirmed through this.
Figure 5 shows the spectrum showing the result of proton analysis using 1H NMR (400 MHz, Chlorform-d) spectroscopy of the compound Q5 of the present invention and the chemical structure confirmed through this.
Figure 6 shows the spectrum showing the result of proton analysis using 1H NMR (400 MHz, Chlorform-d) spectroscopy of Compound A1 of the present invention and the chemical structure confirmed through this.
Figure 7 shows the spectrum showing the result of proton analysis using 1H NMR (400 MHz, Chlorform-d) spectroscopy of Compound A2 of the present invention and the chemical structure confirmed through this.
Figure 8 shows the spectrum showing the result of proton analysis using 1H NMR (400 MHz, Chlorform-d) spectroscopy of Compound A3 of the present invention and the chemical structure confirmed through this.
Figure 9 shows the spectrum showing the result of proton analysis using 1H NMR (400 MHz, Chlorform-d) spectroscopy of Compound A4 of the present invention and the chemical structure confirmed through this.
Figure 10 shows the spectrum showing the result of proton analysis using 1H NMR (400 MHz, Chlorform-d) spectroscopy of the compound Z1 of the present invention and the chemical structure confirmed through this.
Figure 11 shows the spectrum showing the result of proton analysis using 1H NMR (400 MHz, Chlorform-d) spectroscopy of the compound Z2 of the present invention and the chemical structure confirmed through this.
Figure 12 shows the spectrum showing the result of proton analysis using 1H NMR (400 MHz, Chlorform-d) spectroscopy of the compound Z3 of the present invention and the chemical structure confirmed through this spectrum.
13 is a PAGE image showing the result of confirming whether protein labeling is possible according to the treatment of the compound Z3 of the present invention (lane 1: control, lane 2: Z3 (200 μM) without UV, lane 3: Z3 (200 μM) with 365 nm UV).

Definitions of terms used in the present invention are as follows.

Throughout this specification, “include” a certain component means that other components may be further included without excluding other components unless otherwise stated.

In the structural formulas of the present specification, the symbol "-" for bonding atoms and/or substituents (groups) may mean a single bond, the symbol "=" may mean a double bond, and the symbol "≡" may mean a triple bond. The symbol may be omitted, and may be displayed if necessary, such as when specifying a bonding atom or bonding position.

In this specification, "substituted or unsubstituted" may mean that one or a plurality of hydrogen atoms are substituted or unsubstituted with other atoms or substituents unless otherwise specified. Substituents are, for example, halogen, hydroxy, substituted hydroxy, acyloxy, amino, substituted amino, amido, cyano, nitro, guanidino, amidino, mercapto, substituted mercapto, carboxyl, sul phonyloxy, carbonyl, benzyloxy, aryl, heteroaryl, carbocyclyl, heterocyclyl, or other functional groups that may be suitably protected with a protecting group.

As used herein, "alkyl", unless otherwise specified, is a substituted or unsubstituted straight-chain or branched-chain acyclic; cyclic; or a saturated hydrocarbon to which they are bonded. Also, "C _x -C _y alkyl" may mean an alkyl containing x to y carbon atoms. Acyclic alkyl is, for example, methyl, ethyl, N-propyl, N-butyl, N-pentyl, N-hexyl, N-heptyl, N-octyl, isopropyl, sec-butyl, isobutyl , tert-butyl, isopentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like, but is not limited thereto. Cyclic alkyl may include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl, but is not limited thereto. Alkyl in which acyclic and cyclic alkyl are bonded is, for example, methylcyclopropyl, cyclopropylmethyl, ethylcyclopropyl, cyclopropylethyl, methylcyclobutyl, cyclobutylmethyl, ethylcyclopentyl, or cyclopentylmethyl. It may include, but is not limited to.

In this specification, "halogen" may mean fluoro (F), chloro (Cl), bromo (Br) or iodo (I).

As used herein, "carboxyl" means -(C(=0)-OH).

As used herein, “azido” refers to an azide substituent in which three nitrogen atoms are arranged at equal intervals in a straight line, -N ₃ (-N=N ⁺ =N ^- , or -N ^- -N ⁺ ≡N ^- also indicated).

As used herein, "alkyloxycarbonyl" may mean -(C(=O)-O-alkyl), where alkyl is as defined above. In addition, "C ₁ -C ₅ alkyloxycarbonyl" means carbonyloxy containing C ₁ -C ₅ alkyl, that is, -(OC(=O)-(C ₁ -C ₅ alkyl)). It can be, as an example, methylcarbonyloxy (acetoxy, -(OC(=O)-CH ₃ )), ethylcarbonyloxy, n-propylcarbonyloxy, iso-propylcarbonyloxy, n- Butylcarbonyloxy, sec-butylcarbonyloxy, isobutylcarbonyloxy, tert-butylcarbonyloxy, cyclopropylcarbonyloxy, cyclobutylcarbonyloxy, cyclopentylcarbonyloxy, etc. may be included, but It is not limited.

In this specification, "hydroxyalkyl" may mean an alkyl alcohol substituent, ie -(alkyl-OH), where alkyl is as defined above. In addition, "C ₁ -C ₅ hydroxyalkyl" may mean a hydroxy group-bonded C ₁ -C ₅ alkyl, that is, -((C ₁ -C ₅ alkyl) -OH), and one example As , -CH ₂ -OH, -(CH ₂ ) ₂ -OH, -(CH ₂ ) ₃ -OH, -(CH ₂ ) ₄ -OH, and the like may be included, but is not limited thereto.

In the present specification, "amine" is a derivative of ammonia, and unless otherwise specified, may mean a form in which a hydrogen atom is replaced with a substituent such as an alkyl, and the case where one hydrogen is replaced with a substituent in the ammonia Primary amines, when two hydrogens are replaced by substituents are called secondary amines, and when three hydrogens are replaced by substituents are called tertiary amines. As an example, it may include methylamine, dimethylamine, aniline, 3-nitroaniline, and the like, but is not limited thereto.

As used herein, the term "alkynyl" may refer to a substituted or unsubstituted straight-chain, branched-chain, acyclic or cyclic hydrocarbon having at least one carbon-carbon triple bond (C≡C). . In addition, "C ₂ -C ₅ alkynyl" may mean alkynyl containing 2 to 5 carbon atoms, and as an example, 1-propynyl, 2-propynyl (also known as propargyl) ), 1-butynyl, 2-butynyl, and the like, but are not limited thereto.

All technical terms used in the present invention, unless defined otherwise, are used with the same meaning as commonly understood by one of ordinary skill in the art related to the present invention. In addition, although preferred methods or samples are described in this specification, those similar or equivalent thereto are also included in the scope of the present invention. The contents of all publications incorporated herein by reference are incorporated herein by reference.

Hereinafter, the present invention will be described in detail.

In one aspect, the present invention relates to a diazirine-based compound represented by Formula 1 below or a pharmaceutically acceptable salt thereof:

[Formula 1]

(In Formula 1,

R ¹ is halogen, azido or C ₁ -C ₅ alkyloxycarbonyl;

R ² is C ₁ -C ₅ hydroxyalkyl, carboxyl, C ₁ -C ₅ alkyloxycarbonyl, or C ₂ -C ₅ alkynyl;

wherein n is an integer from 1 to 10;

Wherein m is an integer from 0 to 9).

Preferably, R ¹ is halogen, azido or C ₁ -C ₄ alkyloxycarbonyl; R ² is C ₁ -C ₃ hydroxyalkyl, carboxyl, C ₁ -C ₄ alkyloxycarbonyl, or C ₂ -C ₄ alkynyl; wherein n is an integer from 1 to 5; The m may be an integer of 0 to 4.

More preferably, said R ¹ is iodo, azido or -C(=0)-OCH ₂ CH ₃ ; R ² is -CH ₂ OH, carboxyl, -C(=0)-OCH ₂ CH ₃ , or propargyl; wherein n is 1 or 2; The m may be 0 or 1.

Even more preferably, the compound represented by Formula 1 may be one selected from the group of compounds represented by Formulas 2 to 6 below:

[Formula 2]

;

[Formula 3]

;

[Formula 4]

;

[Formula 5]

; and

[Formula 6]

(In Formulas 2 to 6,

wherein n is an integer from 1 to 10; The m may be an integer from 0 to 9).

In Formulas 2 to 5, preferably, n is an integer of 1 to 5; The m may be an integer of 0 to 4, more preferably, the n is 1 or 2; The m may be 0 or 1. In this case, m may be an integer of n-1.

In Formula 6, preferably, n is an integer of 1 to 4; The m may be an integer of 0 to 4, more preferably, the n is 1 or 2; The m may be 0 or 1, and more preferably, the n and m may be 1, respectively.

The compounds represented by Chemical Formulas 2 to 3 may be intermediate compounds for synthesizing the compound represented by Chemical Formula 4.

The compounds represented by Chemical Formulas 2 to 4 may be intermediate compounds for synthesizing the compound represented by Chemical Formula 5.

Particularly preferably, the diazirine-based compound represented by Chemical Formula 1 may be one selected from the group of compounds below:

1) (3-(iodomethyl)-3H-diazirin-3-yl)methanol;

2) (3-(azidomethyl)-3H-diazirin-3-yl)methanol;

3) 3-(azidomethyl)-3H-diazirine-3-carboxylic acid;

4) ethyl 3-(azidomethyl)-3H-diazirine-3-carboxylate;

5) 2-(3-(2-azidoethyl)-3H-diazirin-3-yl)ethan-1-ol; and

6) Ethyl 2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)acetate.

In one embodiment of the present invention, as a result of research to develop diazirine-based compounds that can be useful as photoaffinity label complexes, novel compounds 1 to 6 (compounds Q2, Q3, Q4, Q5, A4 and Z3, see Table 1 below) were synthesized.

compound chemical structure compound name compound chemical structure compound name compound 1
(Q2 of Example 1) (3-(iodomethyl)-3H-diazirin-3-yl)methanol [(3-(iodomethyl)-3H-diazirin-3-yl)methanol] compound 4
(Q5 of Example 4) Ethyl 3-(azidomethyl)-3H-diazirine-3-carboxylate [ethyl 3-(azidomethyl)-3H-diazirine-3-carboxylate] compound 2
(Q3 of Example 2) (3-(azidomethyl)-3H-diazirin-3-yl)methanol [(3-(azidomethyl)-3H-diazirin-3-yl)methanol] compound 5
(A4 of Example 5) 2-(3-(2-azidoethyl)-3H-diazirin-3-yl)acetic acid [2-(3-(2-azidoethyl)-3H-diazirin-3-yl)acetic acid] compound 3
(Q4 of Example 3) 3-(azidomethyl)-3H-diazirine-3-carboxylic acid [3-(azidomethyl)-3H-diazirine-3-carboxylic acid] compound 6
(Z3 of Example 6) Ethyl 2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)acetate [ethyl2-(3-(but-3-yn-1-yl)-3H-diazirin- 3-yl)acetate]

The diazirine-based compound newly synthesized in the present invention binds to a protein and is capable of specific binding with a specific protein and binding between a specific protein and a ligand. The compound of the present invention has a relatively simple structure and a short molecular structure compared to conventional complexes, so the efficiency of action is higher, and it is advantageous when combined with a specific molecule and has the effect of having structural stability. Furthermore, efficient structural changes can be induced, and there is an effect that it is easy to use for the synthesis of new structures. In addition, since the light exposure wavelength of the photoaffinity labeling complex using the compound used in the present invention is 355 nm, the risk to the protein structure is relatively low and the risk to cells is low.

The compound of the present invention can be used in the form of a pharmaceutically acceptable salt, and an acid addition salt formed by a pharmaceutically acceptable free acid is useful as the salt. The term “pharmaceutically acceptable salt” refers to a concentration that has an effective effect that is relatively non-toxic and harmless to patients. It means inorganic addition salt. For these salts, inorganic and organic acids can be used as free acids, hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, perchloric acid, and phosphoric acid can be used as inorganic acids, and citric acid, acetic acid, lactic acid, maleic acid, and fumarin as organic acids. Acids, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, tartaric acid, galacturonic acid, embonic acid, glutamic acid, aspartic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesul For example, phonic acid, 4-toluenesulfonic acid, salicylic acid, citric acid, benzoic acid or malonic acid may be used. In addition, these salts include alkali metal salts (sodium salt, potassium salt, etc.) and alkaline earth metal salts (calcium salt, magnesium salt, etc.) and the like. For example, acid addition salts include acetate, aspartate, benzate, besylate, bicarbonate/carbonate, bisulfate/sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, Gluceptate, gluconate, glucuronate, hexafluorophosphate, hybenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, malic Eight, malonate, mesylate, methyl sulfate, naphthylate, 2-naphsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, saccharide Lates, stearates, succinates, tartrates, tosylates, trifluoroacetates, aluminum, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, Potassium, sodium, tromethamine, zinc salts and the like may be included, of which hydrochloride or trifluoroacetate is preferred.

The acid addition salt according to the present invention is obtained by a conventional method, for example, by dissolving a compound in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile, etc., and adding an organic or inorganic acid to filter and dry the precipitate. It may be prepared by distilling a solvent and excess acid under reduced pressure, followed by drying or crystallization in an organic solvent.

In addition, a pharmaceutically acceptable metal salt may be prepared using a base. An alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare a sodium, potassium or calcium salt as the metal salt. Also, the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate).

Furthermore, the compounds of the present invention include not only the diazirine-based compound represented by Formula 1 and pharmaceutically acceptable salts thereof, but also possible solvates, hydrates, isomers, optical isomers, and the like that can be prepared therefrom.

In one aspect, the present invention is a method for preparing the diazirine-based compound, as shown in Scheme 1 below,

The compound represented by Chemical Formula 7 was reacted by mixing an ammonia solution, and reacted by adding hydroxylamine-O-sulfonicacid and an organic solvent, followed by triethylamine (TEA) and Adding iodine (I ₂ ) and reacting in an organic solvent to obtain a compound represented by Formula 8 (Step 1);

A compound represented by Formula 8 is mixed with imidazole, triphenylphosphine and an organic solvent, and iodine (I ₂ ) is added to react to obtain a compound represented by Formula 2 (Step 2). );

obtaining a compound represented by Chemical Formula 3 by mixing and reacting the compound represented by Chemical Formula 2 with sodium azide and an organic solvent (Step 3);

Obtaining a compound represented by Chemical Formula 4 by reacting a compound represented by Chemical Formula 3 with an organic solvent and a Jones reagent (Step 4); and

Adding magnesium chloride (MgCl ₂ ) and diethylpyrocarbonate to the compound represented by Formula 4 and reacting in an organic solvent to obtain a compound represented by Formula 5 (Step 5);

The diazirine-based compound represented by Chemical Formula 1 is an intermediate compound obtained in Steps 2 to 4 or a final compound obtained in Step 5, wherein the diazirine-based compound represented by Chemical Formula 1 relates to a method for preparing:

[Scheme 1]

(In Scheme 1 above,

wherein n is an integer from 1 to 10;

Wherein m is an integer from 0 to 9).

In Scheme 1, preferably, n is an integer from 1 to 5; The m may be an integer of 0 to 4, more preferably, the n is 1 or 2; The m may be 0 or 1. In this case, m may be an integer of n-1.

Particularly preferably, the compound represented by Formula 1 obtainable by the preparation method as shown in Reaction Scheme 1 may be the following compound:

1) (3-(iodomethyl)-3H-diazirin-3-yl)methanol;

2) (3-(azidomethyl)-3H-diazirin-3-yl)methanol;

3) 3-(azidomethyl)-3H-diazirine-3-carboxylic acid;

4) ethyl 3-(azidomethyl)-3H-diazirine-3-carboxylate; and

5) 2-(3-(2-azidoethyl)-3H-diazirin-3-yl)ethane-1-ol.

In Scheme 1, the organic solvents of steps 1 to 5 are independently methanol (MeOH), ethanol (EtOH), dichloromethane (DCM), dimethylformamide (DMF), acetone, tetrahydrofuran (THF), At least one selected from the group consisting of benzene, KOH/MeOH, MeOH, toluene, hexane, diisopropyl ether, diethyl ether, dioxane, dimethylacetamide (DMA), dimethyl sulfoxide (DMSO) and chlorobenzene can

Preferably, the organic solvent in steps 1 to 5 is independently selected from the group consisting of methanol (MeOH), ethanol (EtOH), dichloromethane (DCM), dimethylformamide (DMF) and acetone. and more preferably, it may be one selected from the group consisting of methanol (MeOH), ethanol (EtOH), dichloromethane (DCM), dimethylformamide (DMF), and acetone.

Especially preferably, the organic solvent of step 1 is methanol (MeOH), the organic solvent of step 2 is dichloromethane (DCM), the organic solvent of step 3 is dimethylformamide (DMF), the organic solvent of step 4 is acetone, step The organic solvent of 5 may be ethanol (EtOH).

In Scheme 1, the steps 1 to 5 independently include drying, evaporating, or removing the solvent/solution after the reaction; filtering; washing; Diluting with water, ethyl acetate (EtOAc) or dichloromethane (DCM); Extracting with ethyl acetate; drying and evaporating the organic layer; and purifying the reactant using column chromatography or the like; Some or all of the steps may be additionally included.

In one aspect, the present invention is a method for preparing the diazirine-based compound, as shown in Scheme 2 below,

As shown in Scheme 2 below,

The compound represented by Chemical Formula 9 is reacted by mixing an ammonia solution, reacted by adding hydroxylamine-O-sulfonicacid and an organic solvent, and then triethylamine (TEA) and Adding iodine (I ₂ ) and reacting in an organic solvent to obtain a compound represented by Formula 10 (Step 1a);

Obtaining a compound represented by Chemical Formula 11 by reacting a compound represented by Chemical Formula 10 with an organic solvent and a Jones reagent (step 4a); and

Adding magnesium chloride (MgCl ₂ ) and diethylpyrocarbonate to the compound represented by Formula 11 and reacting in an organic solvent to obtain a compound represented by Formula 6 (step 5a);

The diazirine-based compound represented by Chemical Formula 1 relates to a method for preparing a diazirine-based compound represented by Chemical Formula 1, wherein the final compound obtained in step 5a is:

[Scheme 2]

(In Scheme 2 above,

wherein n is an integer from 1 to 10;

Wherein m is an integer from 0 to 9).

In Scheme 2, preferably, n is an integer from 1 to 4; The m may be an integer of 0 to 4, more preferably, the n is 1 or 2; The m may be 0 or 1, and more preferably, the n and m may be 1, respectively.

Particularly preferably, the compound represented by Formula 1 obtained by the preparation method as shown in Reaction Scheme 2 may be the following compound:

6) Ethyl 2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)acetate.

In Scheme 2, the organic solvents of step 1a, step 4a and step 5a are independently methanol (MeOH), ethanol (EtOH), dichloromethane (DCM), dimethylformamide (DMF), acetone, tetrahydrofuran ( 1 selected from the group consisting of THF), benzene, KOH/MeOH, MeOH, toluene, hexane, diisopropyl ether, diethyl ether, dioxane, dimethylacetamide (DMA), dimethyl sulfoxide (DMSO) and chlorobenzene There may be more than one species.

Preferably, the organic solvent of step 1a, step 4a and step 5a may be independently one or more selected from the group consisting of methanol (MeOH), ethanol (EtOH) and acetone, more preferably, methanol ( MeOH), it may be one selected from the group consisting of ethanol (EtOH) and acetone.

Especially preferably, the organic solvent of step 1a may be methanol (MeOH), the organic solvent of step 4a may be acetone, and the organic solvent of step 5a may be ethanol (EtOH).

In Scheme 2, steps 1a, 4a, and 5a independently include drying, evaporating, or removing the solvent/solution after the reaction; filtering; washing; Diluting with water, ethyl acetate (EtOAc) or dichloromethane (DCM); Extracting with ethyl acetate; drying and evaporating the organic layer; and purifying the reactant using column chromatography or the like; Some or all of the steps may be additionally included.

In one aspect, the present invention relates to a photo-affinity tag composition comprising the diazirine-based compound represented by Compound 1 or a pharmaceutically acceptable salt thereof.

The photoaffinity labeling composition may label a protein.

The protein may be a polypeptide or amino acid; or derivatives of amino acids (eg, 5-hydroxytryptophan (5-HTP) or L-dihydroxyphenylalanine (L-DOPA)). The amino acids may be natural amino acids or artificial amino acids. The amino acids include glycine, alanine, proline, valine, leucine, isoleucine, methionine, serine, threonine, cysteine, selenocysteine, asparagine, glutamine, phenylalanine, tyrosine, tryptophan, lysine, arginine, histidine, aspartic acid, glutamic acid, It may be selected from the group consisting of ornithine and combinations thereof. In addition, the protein may be a protein in a cell. The cells may be bacterial, plant, or animal cells. The animal cells may be, for example, HEK 293 (Human Embryonic Kidney 293) cells, HeLa, Huh7, Huh7.5, or MDCK cells.

The diazirine-based compound represented by Compound 1 or a pharmaceutically acceptable salt thereof may bind to a protein through a reaction between a protein and a ligand, preferably through a covalent bond, and label the protein (photolabeling).

The diazirine-based compound represented by Compound 1 or a pharmaceutically acceptable salt thereof may be one that labels a protein in response to a light source including ultraviolet (UV) with a wavelength of 200 to 400 nm, and the wavelength of the light is preferably It may be 300 to 380 nm, more preferably 330 to 370 nm. Any light may be used as long as it is emitted from a light source for fluorescence excitation. The light source may be a laser light source. The light may be ultraviolet light.

It is particularly preferable that the diazirine-based compound represented by compound 1 which may be included in the composition for photoaffinity labeling is one selected from the group of compounds below:

4) ethyl 3-(azidomethyl)-3H-diazirine-3-carboxylate;

5) 2-(3-(2-azidoethyl)-3H-diazirin-3-yl)ethan-1-ol; and

6) Ethyl 2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)acetate.

In one aspect, the present invention comprises the steps of treating a protein-containing sample with a diazirine-based compound represented by Compound 1 or a pharmaceutically acceptable salt thereof; And 300 to 380nm wavelength irradiation step of light containing ultraviolet (UV); it relates to a method of photo-labeling a protein, including.

In one aspect, the present invention comprises the steps of treating a protein-containing sample with a diazirine-based compound represented by Compound 1 or a pharmaceutically acceptable salt thereof; And 300 to 380nm wavelength irradiation of light containing ultraviolet (UV); relates to a method for detecting a protein, including.

The protein may be a polypeptide or amino acid; or derivatives of amino acids (eg, 5-hydroxytryptophan (5-HTP) or L-dihydroxyphenylalanine (L-DOPA)). The amino acids may be natural amino acids or artificial amino acids. The amino acids include glycine, alanine, proline, valine, leucine, isoleucine, methionine, serine, threonine, cysteine, selenocysteine, asparagine, glutamine, phenylalanine, tyrosine, tryptophan, lysine, arginine, histidine, aspartic acid, glutamic acid, It may be selected from the group consisting of ornithine and combinations thereof. In addition, the protein may be a protein in a cell. The cells may be bacterial, plant, or animal cells. The animal cells may be, for example, HEK 293 (Human Embryonic Kidney 293) cells, HeLa, Huh7, Huh7.5, or MDCK cells.

The diazirine-based compound represented by Compound 1 or a pharmaceutically acceptable salt thereof may photolabel the protein by binding to the protein through a reaction between the protein and the ligand, preferably through a covalent bond.

In the step of irradiating light, a marker (diazirine-based compound represented by Compound 1 or a pharmaceutically acceptable salt thereof) and a protein may be bound to form a complex between the marker and the protein.

The diazirine-based compound represented by Compound 1 or a pharmaceutically acceptable salt thereof may label a protein in response to the irradiated light, and the wavelength of the light is preferably 300 to 380 nm, more preferably 330 nm. to 370 nm. Any light may be used as long as it is emitted from a light source for fluorescence excitation. The light source may be a laser light source. The light may be ultraviolet light.

It is particularly preferred that the diazirine-based compound represented by Compound 1, which can be used in the method of photo-labeling the protein or the method of detecting the protein, is one selected from the group of compounds below:

4) ethyl 3-(azidomethyl)-3H-diazirine-3-carboxylate;

5) 2-(3-(2-azidoethyl)-3H-diazirin-3-yl)ethan-1-ol; and

6) Ethyl 2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)acetate.

The detection may be performed by measuring fluorescence or absorbance emitted from a complex of a marker (diazirine-based compound represented by Compound 1 or a pharmaceutically acceptable salt thereof) and a protein after light irradiation.

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

<Example 1> Preparation of (3-(iodomethyl)-3H-diazirin-3-yl)methanol (Q2)

<1-1> Preparation of (3H-diazirine-3,3-diyl) dimethanol (Q1)

(3H-diazirine-3,3-diyl) dimethanol [(3H-diazirine-3,3-diyl) dimethanol; In order to synthesize [compound Q1], an ammonia solution was added to a flask containing dihydroxyacetone (compound Q0), sealed at -10 ° C, reacted for 3 hours, and then hydroxyamine-O - After adding 15 ml methanol (MeOH, 15 ml) in which sulfate acid (hydroxylamine-O-sulfonicacid, 2.888 g, 1.15 eq) is dissolved, it was reloaded and mixed and reacted at -10 ° C for 1 hour. After mixing and reacting at room temperature overnight, diazirin was obtained by filtering. The precipitate obtained was filtered, and the filtrate was again concentrated under pressure. After drying, the precipitate was dissolved again in MeOH (40ml), and triethylamine (Et3N; 4.394g, 2eq) was added at 0 °C. After 10 minutes of reaction, I ₂ (6.2 g, 1.1 eq) was added until no decolorization (color change) indicating oxidation occurred to the solution. The mixture was concentrated by air vorbotating under vacuum for 1 hour. The concentrate was diluted with EtOAc (40 mL), washed with saturated Na ₂ S ₂ O ₃ , and the organic layer was dried over Na ₂ SO ₄ and evaporated. The residue was then subjected to silica gel chromatography (EtOAc: Hexane = 3:7, UV) to obtain compound Q1 (650 mg, 47.4%) in the form of a yellow oil.

1H NMR (400 MHz, Chlorform-d) → 3.55 (s, 2H), 2.96 (s, 2H).

<1-2> Preparation of (3-(iodomethyl)-3H-diazirin-3-yl)methanol (Q2)

(3-(iodomethyl)-3H-diazirin-3-yl)methanol [(3-(iodomethyl)-3H-diazirin-3-yl)methanol; For the preparation of [compound Q2], imidazole (511mg, 1.16eg) and triphenylphosphine (Triphenylphosphine, 1.696g, 1eq) were added to compound Q1 (660mg, 1eq) obtained in Example 1-1. DCM (60 mL) was mixed in an ice bath in the dark. After that, iodine powder (1.805 g, 1.1 eq) was slowly added, and then the solution was allowed to come to room temperature by reacting at room temperature overnight. It was diluted again with DCM and washed with saturated Na ₂ S ₂ O ₃ . The organic layer was dried over Na ₂ SO ₄ and evaporated. The residue was purified by silica gel chromatography (EtOAc: Hexane = 3:7, UV) to give Compound Q2 (650 mg, 47.4%) as a yellow oil.

1H NMR (400 MHz, Chlorform-d) → 3.55 (s, 2H), 2.96 (s, 2H).

<Example 2> Preparation of (3-(azidomethyl)-3H-diazirin-3-yl)methanol (Q3)

(3-(azidomethyl)-3H-diazirin-3-yl)methanol [(3-(azidomethyl)-3H-diazirin-3-yl)methanol; For the synthesis of [compound Q3], DMF (8mL) to which sodium azide (520mg, 5eq) was added to Q2 (340mg, 1eq) prepared in the same manner as in Example 1-2 was mixed in a dark room. added. After stirring at room temperature for 12 hours, the mixture was diluted with EtOAc (25 mL) and washed 5 times with brine. The organic layer was dried with Na ₂ SO ₄ and evaporated. The residue was subjected to silica gel chromatography (EtOAc: Hexane = 3:7, PotassiumPermanganate solution as a stain) to obtain Compound Q3 (160 mg, 78.5%) as a yellow oil.

1H NMR (400 MHz, Chlorform-d) → 3.48 (s, 2H), 3.24 (s, 2H).

<Example 3> Preparation of 3-(azidomethyl)-3H-diazirine-3-carboxylic acid (Q4)

3-(azidomethyl)-3H-diazirine-3-carboxylic acid [3-(azidomethyl)-3H-diazirine-3-carboxylic acid; Compound Q4] was synthesized using Q3 prepared by the method of Example 2 and Johnson's reagent.

First, 2.5M Jones reagent was prepared. Specifically, chromium trioxide (2.5 g, 0.025 mol) was put into a 50 mL beaker, dissolved in water (7.5 mL), and then concentrated sulfuric acid (2.5 mL) was slowly added. It was stirred and cooled in an ice water bath. The temperature of the solution was maintained at 0-5 °C. The solution concentration in this step is 2.5M.

Then, 1mL of 2.5M Jones reagent (Chromium trioxide - 3eq) was added with Q3 (100mg, 1eq) dissolved in 0°C acetone (Acetone, 5ml, 1eq) and stirred for 15 minutes. The reaction mixture was stirred at room temperature for 2 hours, and then isopropanol (Isopropyl alcohol, 5 mL) was added and then filtered using an Over Celite method. The filter cake was washed three times with acetone (5 mL) and then dried with Na ₂ SO ₄ . Compound Q4 (50 mg, 45%) was obtained as a colorless oil by filtration, concentration, and purification through silica gel chromatography (Hexane:EtOAc = 7:3, PotassiumPermanganate solution as a stain).

1 H NMR (400 MHz, Chlorform-d) → 1094).

<Example 4> Preparation of ethyl 3-(azidomethyl)-3H-diazirine-3-carboxylate (Q5)

Ethyl 3-(azidomethyl)-3H-diazirine-3-carboxylate [ethyl 3-(azidomethyl)-3H-diazirine-3-carboxylate; Compound Q5] was synthesized using Q4 prepared by the method of Example 4 above. MgCl ₂ (3mg, 0.10eq), diethylpyrocarbonate (Diethylpyrocarbonate, 57mg, 1eq) and Q4 (50mg, 1eq) were put in a vial equipped with a magnetic stirring bar and dissolved in ethyl alcohol (Ethylalcohol, 33mg, 2eq). This mixture was stirred at room temperature for 24 hours. The reaction mixture was diluted with H ₂ O (5 mL) and extracted 3 times with EtOAc (5 mL). The organic layer was separated, dried over Na ₂ SO ₄ , filtered, and the solvent was removed by rotary evaporation. The residue was purified by silica gel chromatography (EtOAc: Hexane = 1:10, PotassiumPermanganate solution as a stain) to give Q5 (13 mg, 21.7%) as a colorless oil.

1H NMR (400 MHz, Chloroform-d) → 4.26 (q, J = 7.1 Hz, 2H), 3.61 (s, 2H), 1.32 (s, 1.32).

<Example 5> Preparation of 2-(3-(2-azidomethyl)-3H-diazirin-3-yl)acetic acid (A4)

<5-1> Preparation of 2,2'-(3H-diazirine-3,3-diyl)bis(ethane-1-ol) (A1)

2,2'-(3H-diazirine-3,3-diyl)bis(ethane-1-ol) [2,2'-(3H-diazirine-3,3-diyl)bis(ethan-1-ol ); To prepare [compound A1], 10 ml of ammonia solution (7M in MeOH) was added to a flask containing dihydroxypentane (1,5-dihydroxypentan-3-1, 350 mg, 1 eq) at -10 ° C. . The flask was sealed and the mixture was mixed at -10 °C for 3 hours. Then, a methanol (2 mL) solution containing hydroxylamine-O-sulfonic acid (hydroxylamine-O-sulfonic acid, 385 mg, 1.15 eq) was added at -10 °C. After sealing the flask and stirring the mixture for 1 hour, the mixture was mixed and reacted overnight at room temperature. A filtrate was obtained through filtration, and the filtrate was concentrated under pressure. The diazirine compound obtained here was dissolved again in methanol (Methanol, 10ml), and Et3N (600mg, 2eq) was added at 0°C. After 10 min, I ₂ (827 mg, 1.1 eq) was added until no decolorization (color change) indicating complete oxidation of the diaziridine intermediate occurred. The mixture was stirred for 1 hour through air vorbotating and then concentrated in vacuo. After that, the mixture was diluted with EtOAc (20 mL) and washed with saturated Na ₂ S ₂ O ₃ . The organic layer was dried over Na ₂ SO ₄ and evaporated. Thereafter, the residue was subjected to silica gel chromatography (EtOAc, Potassium Permanganate solution as a stain) to obtain Compound A1 (237 mg, 62%) in a yellow oil state.

1H NMR (400 MHz, Chlorform-d) → 3.58 (t, J = 5.7 Hz, 4H), 2.62 (s, 2H), 1.69 (t, J = 57.Hz, 4H).

<5-2> Preparation of 2-(3-(2-iodoethyl)-3H-diazirin-3-yl)ethane-1-ol (A2)

2-(3-(2-iodoethyl)-3H-diazirin-3-yl)ethan-1-ol [2-(3-(2-iodoethyl)-3H-diazirin-3-yl)ethan-1 -ol; In order to synthesize [compound A2], imidazole (144mg, 1.16eg) and tryphenylphosphine (Triphenylphosphine, 478mg, 1eq) were added to the compound A1 (237mg, 1eq) obtained by the method of Example 5-1. The added DCM (20 mL) was mixed on an ice bath in the dark. Thereafter, iodine powder (508mg, 1.1eq) was slowly added. The solution was allowed to come to room temperature by reacting overnight at room temperature, then diluted with DCM and washed with saturated Na ₂ S ₂ O ₃ . The organic layer was dried over Na ₂ SO ₄ and evaporated. The residue was subjected to silica gel chromatography (EtOAc: Hexane = 3:7, UV) to give Compound A2 (200 mg, 46%) as a yellow oil.

1H NMR (400 MHz, Chlorform-d) →3.53 (t, J = 6.1 Hz, 2H), 2.94 (t = 7.6 Hz, 2H), 216 (t).

<5-3> Preparation of 2-(3-(2-azidoethyl)-3H-diazirin-3-yl)ethane-1-ol (A3)

2-(3-(2-azidoethyl)-3H-diazirin-3-yl)ethan-1-ol [2-(3-(2-azidoethyl)-3H-diazirin-3-yl)ethan-1 -ol; In order to synthesize [compound A3], DMF (2mL) to which sodium azide (135mg, 5eq) is added is added to A2 (100mg, 1eq) obtained by the method of Example 5-2 in a dark room. did After stirring at room temperature for 12 hours, the mixture was diluted with EtOAc (15 mL) and washed 5 times with brine. The organic layer was dried over Na ₂ SO ₄ and evaporated. The residue was subjected to silica gel chromatography (EtOAc: Hexane = 1:6, PotassiumPermanganate solution as a stain) to obtain Compound A3 (55 mg, 85%) as a colorless oil.

1H NMR (400 MHz, Chlorform-d) → 3.55 (t = 6.0 Hz, 2H), 3.22 (t, J = 6.7 Hz).

<5-4> Preparation of 2-(3-(2-azidoethyl)-3H-diazirin-3-yl)acetic acid (A4)

2-(3-(2-azidoethyl)-3H-diazirin-3-yl)acetic acid [2-(3-(2-azidoethyl)-3H-diazirin-3-yl)acetic acid; Compound A4] was synthesized using A3 obtained by the method of Example 5-3 and Jones reagent.

First, to prepare the 2.5M Jones reagent, Chromiumtrioxide (Chromiumtrioxide, 2.5 g, 0.025 mol) was put in a 50 mL beaker, dissolved in water (7.5 mL), and concentrated sulfuric acid (Sulfonic acid, 2.5 mL) was slowly added. . It was cooled while stirring in an ice water bath, and the temperature of the solution was maintained at 0-5°C. The solution concentration in this step is 2.5M.

Then, for the oxidation of the Jones reagent, 0.5mL of 2.5M Jones reagent (Chromium trioxide - 3eq) was added with A3 (55mg, 1eq) dissolved in 0°C acetone (Acetone, 5ml, 1eq) for 15 minutes. stirred. After stirring the reaction mixture at room temperature for 2 hours, isopropanol (Isopropyl alcohol, 5 mL) was added thereto, and then filtered using an Over Celite method. The filter cake was washed three times with acetone (5 mL) and then dried with Na ₂ SO ₄ . Compound A4 (25 mg, 42%) in a colorless oil state was obtained by filtration, concentration, and purification through silica gel chromatography (Hex/EA = 7/3 + 2% Acetic acid, PotassiumPermanganate solution as a stain).

1 H NMR (400 MHz, Chlorform-d) → 1094).

Calculated MS (ESI) value for [M-H] was 168.06, measured value was 168.05.

<Example 6> Preparation of ethyl 2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)acetate (Z3)

<6-1> Preparation of 2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)ethane-1-ol (Z1)

2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)ethane-1-ol [2-(3-(but-3-yn-1-yl)-3H -diazirin-3-yl)ethan-1-ol; In order to synthesize [compound Z1], an ammonia solution was added to a flask containing 1-hydroxyhept-6-yn-3-1 (1-hydroxyhept-6-yn-3-1, 2g, 1eq). Added, sealed at -10℃ and reacted for 3 hours, then hydroxylamine-O-sulfonic acid (2.06g, 1.15eq) dissolved in 15ml methanol (Methanol, 10ml) was added, and then mixed and reacted at -10 ° C for 1 hour. Then, after mixing at room temperature overnight, diazirin was obtained by filtering, and the obtained precipitate was filtered and the filtrate was concentrated again under pressure. After drying, it was dissolved again in methanol (Methanol, 60ml), and Et3N (3.2g, 2eq) was added at 0 °C. After 10 minutes of reaction, I ₂ (4.43 g, 1.1 eq) was added until no decolorization (color change) indicating oxidation occurred in the solution. The mixture was concentrated by air vorbotating under vacuum for 1 hour. Diluted with EtOAc (70 mL) and washed with saturated Na ₂ S ₂ O ₃ . After washing, the organic layer was dried with Na ₂ SO ₄ and evaporated. Thereafter, the residue was subjected to silica gel chromatography (Hex/EA = 3/2, Potassium Permanganate solution as a stain) to obtain Compound Z1 (900 mg, 41%) as a yellow oil.

1H NMR (400 MHz, Chlorform-d) → 3.53 (q, J = 6.0 Hz, 2H 2) → 2H, 2H).

<6-2> Preparation of 2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)acetic acid (Z2)

2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)acetic acid [2-(3-(but-3-yn-1-yl)-3H-diazirin-3 -yl) acetic acid; Compound Z2] was synthesized using Z1 prepared by the method of Example 6-1 and Jones reagent.

First, for the preparation of 2.5M Jones reagent, Chromiumtrioxide (Chromiumtrioxide, 2.5g, 0.025 mol) was put in a 50 mL beaker, dissolved in water (7.5 mL), and concentrated sulfuric acid (Sulfonic acid, 2.5mL) was slowly added. did It was stirred and cooled in an ice water bath, and the temperature of the solution was maintained at 0-5 °C. The solution concentration in this step is 2.5M.

Then, for the oxidation of the Jones reagent, 4mL of 2.5M Jones reagent (Chromium trioxide - 3eq) was added to Z1 (470mg, 1eq) dissolved in acetone (Acetone, 25ml, 1eq) at 0 ° C for 15 minutes. stirred. The reaction mixture was stirred at room temperature for 2 hours, and then isopropanol (Isopropyl alcohol, 25 mL) was added and then filtered using an Over Celite method. The filter cake was washed three times with acetone (10 mL) and dried with Na ₂ SO ₄ . Compound Z2 (276mg, 53%) was obtained as a colorless oil by filtration, concentration, and purification through silica gel chromatography (Hex/EA= 7/3, PotassiumPermanganatesolutionas a stain).

1H NMR (400MHz, Chlorform-d) →2.44 (s, 2H), 2.10 (td, J = 7.4, 2.6 Hz, 2H), 2.04 (t, J = 2.6 Hz, 1H), 1.83 (t, J = 7.3 Hz, 2H).

<6-3> Preparation of ethyl 2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)acetate (Z3)

Ethyl 2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)acetate [ethyl2-(3-(but-3-yn-1-yl)-3H-diazirin- 3-yl)acetate; In order to synthesize [compound Z3], MgCl ₂ (6mg, 0.10eq), Diethylpyrocarbonate (107mg, 1eq) and Z2 (100mg, 1eq) prepared by the method of Example 6-2 were added to a vial equipped with a magnetic stir bar. It was dissolved with ethyl alcohol (Ethylalcohol, 61mg, 2eq). The mixture was stirred at room temperature for 24 hours, then the reaction mixture was diluted with H ₂ O (2 mL) and extracted three times with EtOAc (3 mL). The organic layer was separated with Na ₂ SO ₄ , dried, filtered, and the solvent was removed by rotary evaporation. The residue was subjected to silica gel chromatography (EtOAc: Hexane = 1:10, PotassiumPermanganate solution as a stain) to give compound Z3 (67mg, 56.7%) as a yellow oil.

1H NMR (400MHz, Chloroform-d) → 4.22 (q, J = 7.1 Hz, 2H), 2.36 (s, 2H), 2.09 (td, J = 7.4, 2.6 Hz, 2H), 2.02 (t, J = 2.7 Hz, 1H), 1.81 (t, J = 7.4 Hz, 2H), 1.32 (t, J = 7.2 Hz, 3H).

<Example 7> Labeling of proteins using the compounds of Examples

In order to confirm the usefulness of the compound obtained by the method of the examples of the present invention as a photo-affinity tag, a cell lysate sample obtained by lysing Huh7.5 cells was performed. After treatment with compound Z3 of Example 6 at a concentration of 200 µM and/or irradiation with ultraviolet (UV) light at a wavelength of 365 nm, SDS-PAGE was performed and protein labeling was analyzed. At this time, lane 1 is a control group treated with Z3 and irradiated with UV, lane 2 is a group treated with Z3 but not irradiated with UV, lane 3 is a group treated with Z3 and irradiated with UV .

As a result of the analysis, it was confirmed that Huh7.5 cell lysate was efficiently labeled through photocrosslinking with proteins only when Z3 (200 μM) was treated and then 365 nm wavelength light was treated (lane 3) (FIG. 13) .

In addition, even in the case of Compound Q5 of Example 4 and Compound A4 of Example 5, it was confirmed that proteins can be efficiently labeled when irradiated with light having a wavelength of 365 nm.

Therefore, from these results, it was confirmed that the compounds prepared by the methods of Examples 1 to 6 of the present invention can be usefully used as compounds for photoaffinity labeling capable of labeling a wide variety of proteins.

So far, the present invention has been looked at with respect to its preferred embodiments. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting point of view. The scope of the present invention is particularly indicated in the claims rather than the foregoing description, and all differences within the equivalent range should be construed as being included in the present invention.

Claims (12)

Diazirine-based compound represented by Formula 1 below or a pharmaceutically acceptable salt thereof:
[Formula 1]

(In Formula 1,
R ¹ is halogen, azido or C ₁ -C ₅ alkyloxycarbonyl;
R ² is C ₁ -C ₅ hydroxyalkyl, carboxyl, C ₁ -C ₅ alkyloxycarbonyl, or C ₂ -C ₅ alkynyl;
wherein n is an integer from 1 to 10;
Wherein m is an integer from 0 to 9). According to claim 1,
R ¹ is iodo, azido or -C(=0)-OCH ₂ CH ₃ ;
R ² is -CH ₂ OH, carboxyl, -C(=0)-OCH ₂ CH ₃ , or propargyl;
wherein n is 1 or 2;
Wherein m is 0 or 1, a diazirine-based compound or a pharmaceutically acceptable salt thereof. According to claim 1,
The compound represented by Formula 1 is one selected from the group of compounds represented by Formulas 2 to 6 below, a diazirine-based compound or a pharmaceutically acceptable salt thereof:
[Formula 2]
;
[Formula 3]
;
[Formula 4]
;
[Formula 5]
; and
[Formula 6]

(In Formulas 2 to 6,
The n and m are each as defined in claim 1). According to claim 3,
A diazirine-based compound or a pharmaceutically acceptable salt thereof, characterized in that the compound represented by Chemical Formulas 2 to 3 is an intermediate compound for synthesizing the compound represented by Chemical Formula 4. According to claim 3,
A diazirine-based compound or a pharmaceutically acceptable salt thereof, characterized in that the compounds represented by Chemical Formulas 2 to 4 are intermediate compounds for synthesizing the compound represented by Chemical Formula 5. According to claim 1,
The diazirine-based compound represented by Formula 1 is one selected from the group of compounds below, characterized in that, a diazirine-based compound or a pharmaceutically acceptable salt thereof:
1) (3-(iodomethyl)-3H-diazirin-3-yl)methanol;
2) (3-(azidomethyl)-3H-diazirin-3-yl)methanol;
3) 3-(azidomethyl)-3H-diazirine-3-carboxylic acid;
4) ethyl 3-(azidomethyl)-3H-diazirine-3-carboxylate;
5) 2-(3-(2-azidoethyl)-3H-diazirin-3-yl)ethan-1-ol; and
6) Ethyl 2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)acetate. A method for producing the diazirine-based compound represented by Formula 1 of claim 1,
As shown in Scheme 1 below,
The compound represented by Formula 7 was reacted by mixing an ammonia solution, and reacted by adding hydroxylamine-O-sulfonicacid and an organic solvent, followed by triethylamine (TEA) and Adding iodine (I ₂ ) and reacting in an organic solvent to obtain a compound represented by Formula 8 (Step 1);
A compound represented by Formula 8 is mixed with imidazole, triphenylphosphine and an organic solvent, and iodine (I ₂ ) is added to react to obtain a compound represented by Formula 2 (Step 2). );
obtaining a compound represented by Chemical Formula 3 by mixing and reacting the compound represented by Chemical Formula 2 with sodium azide and an organic solvent (Step 3);
Obtaining a compound represented by Chemical Formula 4 by reacting a compound represented by Chemical Formula 3 with an organic solvent and a Jones reagent (Step 4); and
Adding magnesium chloride (MgCl ₂ ) and diethylpyrocarbonate to the compound represented by Formula 4 and reacting in an organic solvent to obtain a compound represented by Formula 5 (Step 5);
The method for producing a diazirine-based compound represented by Chemical Formula 1 of claim 1, wherein the diazirine-based compound represented by Chemical Formula 1 is an intermediate compound obtained in Steps 2 to 4 or a final compound obtained in Step 5:
[Scheme 1]

(In Scheme 1 above,
The n and m are each as defined in claim 1). According to claim 7,
The organic solvents of steps 1 to 5 are independently methanol (MeOH), ethanol (EtOH), dichloromethane (DCM), dimethylformamide (DMF), acetone, tetrahydrofuran (THF), benzene, KOH / MeOH, MeOH, toluene, hexane, diisopropyl ether, diethyl ether, dioxane, dimethylacetamide (DMA), dimethyl sulfoxide (DMSO) and at least one selected from the group consisting of chlorobenzene, the production method. A method for producing the diazirine-based compound represented by Formula 1 of claim 1,
As shown in Scheme 2 below,
The compound represented by Chemical Formula 9 is reacted by mixing an ammonia solution, reacted by adding hydroxylamine-O-sulfonicacid and an organic solvent, and then triethylamine (TEA) and Adding iodine (I ₂ ) and reacting in an organic solvent to obtain a compound represented by Formula 10 (Step 1a);
Obtaining a compound represented by Chemical Formula 11 by reacting a compound represented by Chemical Formula 10 with an organic solvent and a Jones reagent (step 4a); and
Adding magnesium chloride (MgCl ₂ ) and diethylpyrocarbonate to the compound represented by Formula 11 and reacting in an organic solvent to obtain a compound represented by Formula 6 (step 5a);
Method for preparing a diazirine-based compound represented by Chemical Formula 1 of claim 1, wherein the diazirine-based compound represented by Chemical Formula 1 is the final compound obtained in step 5a:
[Scheme 2]

(In Scheme 2 above,
The n and m are each as defined in claim 1). According to claim 9,
The organic solvents of step 1a, step 4a and step 5a are independently methanol (MeOH), ethanol (EtOH), dichloromethane (DCM), dimethylformamide (DMF), acetone, tetrahydrofuran (THF), benzene, KOH / MeOH, MeOH, toluene, hexane, diisopropyl ether, diethyl ether, dioxane, dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), and at least one selected from the group consisting of chlorobenzene, prepared method. A composition for a photo-affinity tag comprising the diazirine-based compound represented by compound 1 of claim 1 or a pharmaceutically acceptable salt thereof. Treating a protein-containing sample with a diazirine-based compound or a pharmaceutically acceptable salt thereof represented by Compound 1 of claim 1; and
300 to 380nm wavelength irradiation of light containing ultraviolet (UV); containing, a method of photolabeling a protein.