JP4122446B2 - Phenyldiazirine-added nucleic acid derivative and production method thereof, phenyldiazirine-added nucleotide derivative and production method thereof, and protein analysis method and preparation method - Google Patents

Description

The present invention is phenyl diazirine additional nucleic acid derivatives and process for producing the same, and phenyl diazirine additional nucleotide derivative and its production method. The present invention relates to analytical methods and preparation methods of proteins using phenyl diazirine additional nucleic acid derivatives and phenyl diazirine additional nucleotide derivatives.

  Nucleic acids are basic substances of life and are responsible for many functions of life phenomena. A single nucleic acid and its derivative are responsible for various information transmissions, energy transmissions, and the like in a living body. Nucleic acid complexes, that is, DNA and RNA are basic compounds that support life and have blueprints and protein production functions. The reason why nucleic acids and their derivatives and complexes can exert various functions in this way is because there are proteins that bind to them and work. ("Molecular Biology of Cells, 4th Edition" by Bruce Alberts, translated by Keiko Nakamura and Kenichi Matsubara, published by Newton Press (Non-patent Document 1))

  Protein motif analysis is often used as a method for analyzing proteins that bind to nucleic acids and their derivatives. The site of the protein that binds to the nucleic acid and its derivative is somewhat similar to each other, and this property is used to predict whether the protein will bind to the nucleic acid and its derivative.

  As a method for analyzing a protein that binds to a nucleic acid complex, DNA, or RNA, an electric mobility shift assay (hereinafter, EMSA) is often used. EMSA detects a protein that binds to a DNA or RNA fragment having a specific sequence by the following method. A nuclear extract containing a large amount of DNA or RNA-binding protein and a DNA or RNA fragment having a specific sequence labeled for detection are mixed, and a complex is formed with mutual affinity while being placed for a certain period of time. Let Thereafter, DNA or RNA that has not bound to the DNA or RNA-protein complex is separated by non-denaturing electrophoresis, and the labeled DNA or RNA is visualized by an appropriate detection method. At this time, the complex generally has a slower mobility during electrophoresis than the DNA or RNA alone. (Molecular Cloning A Laboratory Manual 3rd Edition, Sambrook J, Russell DW, pages 17-13-17-22, published by Cold Spring Harbor Laboratory Press (Non-patent Document 2))

  The present inventors have so far developed an original high-speed photoaffinity method using a diazirine derivative as a photoreactive group. The photoaffinity method is a technique for irreversibly linking a specific ligand and a partner protein using a photoreaction. Diazirine has the advantage of favoring this irreversible binding compared to other photoreactive groups. (Y. Sadakane, Y. Hatanaka (2004) Multifunctional photoprobes for rapid protein identification. In F. Darvas, A. Guttman and G. Dorman eds., Chemical Genomics, Marcel Dekker Inc., New York. Pp199-214) Reference 3), Y. Hatanaka, Y. Sadakane (2002) Photoaffinity labeling in drug discovery and developments: Chemical gateway for entering proteomic frontier. Curr. Top. Med. Chem. 2, 271-288 (Non-patent Document 4), Hatanaka Homaru Structural Biological Organic Chemistry: Probing Protein Functional Structures with Photoaffinity Labels, Journal of Organic Synthesis, 56 (7), 581-590, 1998 (Non-patent Document 5), M. Kaneda, Y. Sadakane, Y Hatanaka, (2003) A Novel Approach for Affinity-Based Screening of Target Specific Ligands: Application of Photoreactive D-Glyceraldehyde-3-phosphate Dehydrogenase. Bioconjugate Chem. 14, 849-852. (Non-Patent Document 6), M. Hashimoto , J. Yang, Y. Hatanaka, Y. Sadakane, K. Nakagomi, GD Holman (2002) Improvement in the properties of 3-phen yl-3-trifluoromethyldiazirine based photoreactive bis-glucose probes for GLUT4 following substitution on the phenyl ring. Chem. Pharm. Bull. 50, 1004-1006 (Non-patent Document 7), Hatanaka, Y., Kempin, U., Park, JJ. One-step synthesis of biotinyl photoprobes from unprotected carbohydrates. J. Org. Chem. 65, 5639-5643, 2000 (Non-Patent Document 8), Hatanaka, Y., Hashimoto, H., Kanaoka, Y. A rapid and J. Am. Chem. Soc. 120, 453-454, 1998 (Non-patent Document 9), Japanese Patent Laid-Open No. 2000-319262 (Patent Document 1)) efficient method for identifying photoaffinity biotinylated sites within proteins.

The following problems remain unsolved in analyzing, separating and purifying nucleic acids and their derivatives and their complexes.
(1) The method of analyzing a protein to which a nucleic acid and its derivative are bound from a homologous motif is not a direct confirmation method, and cannot be used as a method for searching for a novel binding protein whose amino acid sequence is unknown.
(2) EMSA, which is frequently used for analysis of binding proteins of DNA or RNA, has a very poor resolution due to the restriction of non-denaturing electrophoresis, and is an analysis method that only confirms the presence or absence of binding proteins. For example, even when there are a plurality of binding proteins in the analysis system, EMSA can only obtain information on the presence or absence of binding proteins, and cannot know the number of proteins. It is well known that a plurality of proteins form higher-order complexes and bind to DNA or RNA, and the limit of EMSA has been pointed out as an analysis method.
(3) A general method for separating and purifying a protein that binds to a nucleic acid, a derivative thereof and a complex thereof from a protein mixture has not yet been established.

  Accordingly, the present invention provides a method for producing a photoreactive nucleic acid derivative or nucleotide derivative for the purpose of solving the above-mentioned problems, and further provides a comprehensive protein analysis method using the produced derivative. It is an object of the present invention to provide a method for isolating a binding protein (a method for producing a protein).

The present invention for solving the above problems is as follows.
[1]
Reacting a nucleic acid derivative which is a compound represented by the general formula (A) with a phenyldiazirine compound having a reactive group with a thiol group of a thiophosphate group,
Ri compounds der said phenyldiazirine compound represented by the general formula (C), and
A method for producing a phenyldiazirine-added nucleic acid derivative, wherein the phenyldiazirine-added nucleic acid derivative is a compound represented by the general formula (D) .
In general formula (C), R ₅ is
R ₆ is a halogen atom;
R ₇ is an alkylsulfonyl group having 1 to 6 carbon atoms ,
R ₈ is H;
n is an integer of 1-6.
In general formula (A), x and y are each independently an integer of 0 to 100, and N is a group represented by the following general formula (B).
In general formula (B), R ₁ is an OH group or an adjacent nucleotide, R ₂ is a hydrogen or OH group, R ₃ is an OH group or an adjacent nucleotide, and B is adenine, guanine, cytosine, uracil. , Thymine, 5-methylcytosine, N6-methyladenine, 5-hydroxymethylcytosine, and inosine, and X is S (sulfur) or O (oxygen).
In general formula (D), x and y and N are the same as defined in general formula (A), and R ₄ is
And n is an integer of 1-6.
[ 2 ]
The method according to [1], wherein the phenyldiazirine compound represented by the general formula (C) is a compound represented by the following formulas (1) to ( 3 ).

[ 3 ]
The production method according to any one of [1] to [ 2 ], wherein the nucleic acid derivative and / or the phenyldiazirine-added nucleic acid derivative has a label.
[ 4 ]
Label, biotin, a manufacturing method according to the radioisotope, is a fluorescent substance was or [3].
[ 5 ]
A phenyldiazirine-added nucleic acid derivative represented by the general formula (D).
In general formula (D), R ₄ is
N is an integer of 1 to 6, x and y are independently an integer of 0 to 100, and N is a group represented by the following general formula (B).
In general formula (B), R ₁ is an OH group or an adjacent nucleotide, R ₂ is a hydrogen or OH group, R ₃ is an OH group or an adjacent nucleotide, and B is adenine, guanine, cytosine, uracil. , thymine, and 5-methylcytosine, N6-methyl adenine, 5-hydroxymethyl cytosine, and salts group Ru is selected from the group consisting of inosine, X is S (sulfur) or O (oxygen).
[ 6 ]
The derivative according to [ 5 ], wherein the phenyldiazirine-added nucleic acid derivative has a label.
[ 7 ]
Label, biotin, derivative according to radioisotopes, it is a fluorescent substance was or [6].
[ 8 ]
Reacting a nucleotide derivative represented by the following general formula (E) or (F) with a phenyldiazirine compound having a reactive group with a thiol group in the compound represented by the general formula (E) or (F) And the phenyldiazirine compound is a compound represented by the general formula (C).
In the general formula (E), R is an adenine, guanine, cytosine, uracil, thymine, 5-methylcytosine, N6-methyl adenine, 5-hydroxymethyl cytosine, and salts group Ru is selected from the group consisting of inosine, n Is 0, 1 or 2, and in general formula (F), R ₁ and R ₂ are independently nicotinamide adenine nucleotide and its phosphate, and flavin mononucleotide, sugar phosphate, sugar nucleotide, coenzyme A, and selected from the group consisting of phospholipids.
In general formula (C), R ₅ is
R ₆ is a halogen atom;
R ₇ is an alkylsulfonyl group having 1 to 6 carbon atoms ,
R ₈ is H;
n is an integer of 1-6.
[ 9 ]
The method according to [ 8 ], wherein the phenyldiazirine compound is a compound represented by the following formulas (1) to ( 3 ).
[ 10 ]
The phenyldiazirine-added nucleic acid derivative produced by the method according to any one of [1] to [ 4 ], the phenyldiazirine-added nucleic acid derivative according to any of [ 5 ] to [ 7 ], or [ 8 ] to [ 8 ] 9 ] the nucleotide derivative produced by the method according to any one of the above and the protein as the analyte are mixed under conditions that allow interaction,
The obtained mixture is irradiated with light to react the diazirine group contained in the phenyldiazirine-added nucleic acid derivative or nucleotide derivative with the protein, and to combine the phenyldiazirine-added nucleic acid derivative or nucleotide derivative with the protein. Forming and then subjecting the resulting conjugate to electrophoresis,
Protein analysis method by electrical mobility shift assay.
[ 11 ]
The method according to [ 10 ], wherein the electrophoresis is performed under denaturing conditions.
[ 12 ]
[1] phenyldiazirine additional nucleic acid derivatives prepared by the method of any crab according to [4], [5] phenyldiazirine additional nucleic acid derivative of any crab according to [7] or [8], - [ 9 ] the nucleotide derivative produced by the method according to any one of the above and the protein as the analyte are mixed under conditions that allow interaction,
The obtained mixture is irradiated with light to react the diazirine group contained in the phenyldiazirine-added nucleic acid derivative or nucleotide derivative with the protein, and to combine the phenyldiazirine-added nucleic acid derivative or nucleotide derivative with the protein. Forming,
The resulting conjugate is subjected to electrophoresis,
Separating the conjugate, and then treating the separated conjugate with an alkaline solution to dissociate the conjugate;
Recovering the dissociated protein and / or nucleic acid derivative or nucleotide derivative,
Protein preparation method.
[ 13 ]
The method according to [ 12 ], wherein the electrophoresis is performed under denaturing conditions.

  According to the present invention, photoreactive nucleic acid derivatives and nucleotide derivatives can be provided. By using these derivatives, it is possible to capture proteins that bind to them by covalent bonds. This photoreaction is efficient and extremely fast with light near 360 nm. Further, this light is hardly absorbed by normal biomolecules such as nucleic acids such as DNA and proteins. Therefore, a complex of a photoreactive compound (nucleic acid derivative) and a binding protein can be applied to most existing protein analysis methods. This makes it possible to comprehensively analyze a plurality of binding proteins.

  Furthermore, the photoreactive group in the photoreactive compound can be separated from the nucleic acid. Using this cleavage reaction, only the binding protein is extracted from the complex of the photoreactive compound (nucleic acid derivative) and the binding protein. It becomes possible.

[Method for producing phenyldiazirine- added nucleic acid derivative (1)]
The method (1) for producing a phenyldiazirine- added nucleic acid derivative of the present invention comprises a nucleic acid derivative in which the phosphate group of at least one nucleic acid is a thiophosphate group, a phenyldiazirine compound having a reactive group with a thiol group of the thiophosphate group, Reaction.

The nucleic acid derivative in which the phosphate group of at least one nucleic acid is a thiophosphate group can be, for example, a compound represented by the general formula (A).

  In general formula (A), x and y are each independently an integer of 0 to 100. The sum of x and y is 0 or more and 200 or less. However, the production method of the present invention can also be applied to nucleic acid derivatives in which the sum of x and y exceeds 200.

In general formula (A), N is a group represented by the following general formula (B).

In general formula (B), R ₁ is an OH group or an adjacent nucleotide. When the corresponding N is the terminal of the nucleic acid derivative, R ₁ is an OH group, otherwise R ₁ is the adjacent nucleotide (N). R ₂ is hydrogen or an OH group. When R ₂ is hydrogen, the nucleic acid derivative is DNA, and when R ₂ is an OH group, the nucleic acid derivative is RNA. In one nucleic acid derivative, a nucleotide (N) in which R ₂ is hydrogen and a nucleotide (N) in which R ₂ is an OH group can be mixed. R ₃ is an OH group or an adjacent nucleotide. When the corresponding N is the terminal of the nucleic acid derivative, R ₃ is an OH group, otherwise R ₃ is the adjacent nucleotide (N). B is adenine, guanine, cytosine, uracil, thymine base and base derivatives. Examples of the base derivative may include, but are not limited to, 5-methylcytosine, N6-methyladenine, 5-hydroxymethylcytosine, inosine, and the like. B is independently selected from the above bases independently for each nucleotide (N), and can constitute nucleic acid derivatives having various base sequences.

X is S (sulfur) or O (oxygen). X in normal nucleotide (N) is O (oxygen) and constitutes a phosphate group. The phosphate group in which X is S (sulfur) is a thiophosphate group. In the general formula (A), the nucleotide (N) represented by N-SH displays SH of the thiophosphate group in the general formula (A). The nucleotide (N) other than N-SH may also contain a nucleotide (N) in which X is S (sulfur). In the nucleotide (N) where X is S (sulfur), a phenyldiazirine group can be introduced by the method of the present invention, and when one nucleic acid derivative has a plurality of thiophosphate groups, It is possible to introduce a phenyldiazirine group.

The nucleic acid derivative can have a label. The label can be a known label used for nucleic acid derivatives. Such labels can include, for example, biotin, radioisotopes, and / or fluorescent materials. However, it is not limited to these substances. Introduction of a label into a nucleic acid derivative can be performed by a known method described in the following document.
S. Agrawal; C. Christodoulou; MJ Gait (1986) Efficient methods for attaching non-radioactive labels to the 5 'ends of synthetic oligodeoxyribonucleotides Nucleic Acids Res. 14: 6227-6245. "Nonisotopic DNA probe techniques" (ed. LJ Kricka ) Academic Press, New York

  Among DNA and RNA, those having a thiophosphate group in which X is S (sulfur) are known and can be appropriately synthesized by known methods described in the following documents. Zon, G .; Geiser, TG (1991) Phosphorothioate oligonucleotides: chemistry, purification, analysis, scale-up and future directions.Anticancer Drug Des. 6: 539-568. And Caruthers, MH; Beaton, G .; Wu, JV ; Wiesler, W. (1992) Chemical synthesis of deoxyoligonucleotides and deoxyoligonucleotide analogs.Methods Enzymol. 211: 3-20.

Phenyl diazirine compound can be, for example, a compound represented by the general formula (C).

In general formula (C), R ₅ is

In the above formula, R ₆ is a halogen atom (eg, Cl, Br, I), or a sulfonic acid ester residue. Specifically, the sulfonic acid ester residue can be a methanesulfonyl group or a toluenesulfonyl group. R ₇ is an alkylthiosulfonyl group having 1 to 6 carbon atoms or an alkylthio group having 1 to 6 carbon atoms. Alkylthio alkylsulfonyl group, RSO ₂ - indicated, R is for example, be methyl, ethyl, propyl or butyl. The alkylthio group is represented by RS- and R can be, for example, methyl, ethyl, propyl or butyl. R ₈ can be H or an alkyl group having 1 to 6 carbon atoms, and the alkyl group can be, for example, methyl, ethyl, propyl, or butyl. n is an integer of 1 to 6, preferably an integer of 1 to 4, and more preferably 1.

The phenyldiazirine compound represented by the general formula (C) can be, for example, a compound represented by the following formula (1).

The compound represented by the formula (1) has a maleimide group and diazirine as reactive functional groups. The maleimide group reacts with the thiol group of the thiophosphate group of the nucleic acid derivative having a thiophosphate group. The compound represented by the formula (1) can be produced by a reaction with the phenyldiazirine compound of the formula (2) or the formula (7).

The phenyldiazirine compound represented by the general formula (C) can be, for example, a compound represented by the following formula (2).

  The compound represented by the formula (2) has halogen (Hal) and diazirine as reactive functional groups. Halogen (Hal) reacts with the thiol group of the thiophosphate group of the nucleic acid derivative having a thiophosphate group. Examples of halogen (Hal) include Cl, Br, and I. The compound represented by the formula (2) can be produced by a method described in the literature [M. Nassal, J. Am. Chem. Soc., 106, 7540-7545 (1984); LB Shih, and H Bayley, Anal. Biochem., 1985, 144, 132-141].

  In an existing synthesis method, a functional group that is a key at the time of introduction into a ligand is previously incorporated on a phenyl skeleton, and a diazirine skeleton that is a photoreactive group is constructed. For example, the halogenated derivatives (2a) and (2b) of phenyldiazirine shown in Scheme 1 below are extremely useful intermediates for the synthesis of various photoreactive probes. In the synthesis methods reported so far, all the functional groups necessary for halogenation are synthesized in advance because they are assembled in advance before the construction of the diazirine skeleton.

The phenyldiazirine compound represented by the general formula (C) can be, for example, a compound represented by the following formula (3).

  The compound represented by the formula (3) is a non-patent document 6, M. Kaneda, Y. Sadakane, Y. Hatanaka, (2003) A Novel Approach for Affinity-Based Screening of Target Specific Ligands: Application of Photoreactive D-Glyceraldehyde. -3-phosphate Dehydrogenase. Bioconjugate Chem. 14, 849-852.

The phenyldiazirine compound represented by the general formula (C) can be, for example, compounds represented by the following formulas (4) to (10).

  In formulas (4), (5), (6), and (9), R is hydrogen (H), an unsubstituted or substituted alkyl group having 1 to 6 carbon atoms, and in formula (10), R is , Hydrogen (H), an unsubstituted or substituted alkyl group having 1 to 6 carbon atoms, and R ′ is H; an unsubstituted or substituted alkylcarbonyl group having 2 to 6 carbon atoms such as an acetyl group; tert-butoxycarbonyl (Boc ) Group, an unsubstituted or substituted alkylcarbonyloxy group having 2 to 6 carbon atoms such as 9-fluorenylmethoxycarbonyl (Fmoc) group; or an arylcarbonyloxy group such as benzoyl group, Is an alkanesulfonyl group such as methanesulfonyl or a benzenesulfonyl group such as p-toluenesulfonyl. The unsubstituted alkyl group having 1 to 6 carbon atoms represented by R is, for example, methyl, ethyl, n-propyl, or t-butyl, and the substituted alkyl group is, for example, benzyl.

  As shown in the following scheme 2, the phenyldiazirine derivatives represented by the formulas (4) to (10) can be synthesized in the simplest and most large amount by using unsubstituted phenyldiazirine (11) as a starting material ( 11) The intermediate (12) in which the aldehyde group is directly modified is synthesized, and the aldehyde group of this intermediate (12) is further modified to obtain the phenyldiazirine derivative represented by the formulas (4) to (10). Can be synthesized.

[Direct modification of phenyldiazirine]
Phenyldiazirine is known as an excellent photoreactive group because it is stable to various synthesis conditions, but can be rapidly decomposed by photoreaction to form a stable crosslink. However, in order to introduce this into a chemical substance or biomolecule, an appropriate functional group is required, and the synthetic complexity at that stage has been a problem. First, we developed a method for easily synthesizing a useful intermediate aldehyde (12) using a large amount of synthesizable (11) as a raw material, and pioneered a new route through which various useful diazirines can be easily derived. As for (12), a synthesis method with poor yield which has passed through 7 steps has been reported so far [JM Delfino, SL Schreiber, and FM Richards, J. Am. Chem. Soc., 1993, 115, 3458-3474] . However, in the present invention, instead of this synthetic method, a completely new approach to obtain aldehyde (12) in one step by directly modifying phenyldiazirine (11) is used (Scheme 3).

Until now, by reacting phenyldiazirine with Cl ₂ CHOCH ₃ in the presence of GaCl _{3 in} TFA-CH ₂ Cl ₂ , the aldehyde (12) was successfully obtained, although the yield was only 5%. [U. Kempin, Y. Kanaoka, and Y. Hatanaka, Heterocycles, 1988, 49, 465-468.]. During this experiment, it was found that the raw material Cl ₂ CHOCH ₃ decomposed immediately after the reaction started. As a result of investigating the cause, decomposition occurs when Cl ₂ CHOCH ₃ is added to TFA. As a result of examining an organic acid that replaces TFA, Cl ₂ CHOCH ₃ is found to be stable in trifluoromethanesulfonic acid (TfOH). I understood. Under these conditions, Lewis acid is AlCl ₃ , ZnCl ₂ , SbCl ₃ , SbCl ₅ , SnCl ₂ , SnCl ₄ , Zn (OTf) ₂ , AgOTf, Gd (OTf) ₃ , Pr (OTf) ₃ , Sm (OTf) ₃ , Y (OTf) ₃ , Yb (OTf) ₃ , and other various Lewis acids were examined. The formation of the desired aldehyde (12) was confirmed on TLC. Among these, the yields for GaCl ₃ , FeCl ₃ , SbF ₅ , TiCl ₄ are shown in Table 1.

  This method has made it possible to easily introduce a functional group useful for synthesis onto the aromatic ring of phenyldiazirine, which has been difficult to modify directly until now, and the obtained aldehyde has various functional groups. It is considered to be particularly important in terms of being easily convertible to

[Application to simple asymmetric synthesis of phenylalanine derivatives with diazirine groups]
Aldehydes (12), which can be easily obtained by Friedel-Crafts reaction, are further reduced to various useful synthetic intermediates such as benzyl alcohol (13), hydroxy group halogenated compound (14), etc. Can be easily converted into a body. This halogen compound is used for the synthesis of phenylalanine (Tmd (Phe)) (15) having a diazirine group at the p-position, which is useful for the synthesis of photoreactive peptides and proteins [for example, M. Nassal, J. Am. Chem. Soc., 106, 7540-7545 (1984); LB Shih, and H. Bayley, Anal. Biochem., 1985, 144, 132-141C; WG Fishwick, JM Sanderson, and JBC Findlay, Tetrahedron Lett., 1994, 35, 4611-4614.] This is a very multi-step synthesis method involving diazirine synthesis. Here, starting from the aldehyde form (12), reduction gives (13), followed by halogenation to give (14). (14) to (15) can be easily asymmetrically synthesized in several steps (Scheme 4). In the following scheme 4, (13) is the same compound as the compound (7), (14) is a compound in which Hal is Br in the compound (2), and (15) is the above (10 ), R and R ′ are hydrogen atoms (H).

Reaction of Nucleic Acid Derivative with Phenyldiazirine Compound Having Thiol-Reactive Group In the method (1) of the present invention, a phenyldiazirine compound having a thiol-reactive group and a nucleic acid derivative are, for example, about 50: 1 to 100: 1 In a molar ratio. Furthermore, diisopropylethylamine in a molar ratio of 50 to 100 times with respect to the nucleic acid derivative is added to this mixture, and the reaction can be carried out in dimethyl sulfoxide at 37 ° C. However, N-methylmorpholine, triethylamine or the like can be used instead of diisopropylethylamine. As the reaction solvent, methanol, dimethylformamide or the like can be used instead of dimethyl sulfoxide. The reaction temperature can be in the range of, for example, 4 to 70 ° C., including 37 ° C.

The concentration of the phenyldiazirine compound is suitably in the range of 0.1 to 0.5 mM, for example. The reaction time depends on the type of the phenyldiazirine compound, but can be, for example, about several tens of minutes to several hours. It is preferable to store in liquid nitrogen after the reaction so as not to cause excessive reaction. The product phenyldiazirine- added nucleic acid derivative can be purified by, for example, high performance liquid chromatography. In addition to high performance liquid chromatography, it can also be purified by, for example, capillary electrophoresis.

Examples of the above reaction are shown in the scheme below.
Scheme 5 is an example in which the compound (2a) is used as the phenyldiazirine compound. The nucleic acid derivative (A1) is an example having a thiophosphate group at a nucleotide other than the terminal nucleotide of the nucleic acid derivative. Hydrogen with a compound of the thiol thiophosphate group and Br of (2a) forms a HBr, by de-HBr, compound (2a) with a nucleic acid derivative (A1) and is attached, a phenyl diazirine additional nucleic acid derivative (D1) Generate.

Scheme 6 is an example in which the compound (3) is used as a phenyldiazirine compound. The nucleic acid derivative (A2) is an example having a thiophosphate group at the terminal nucleotide of the nucleic acid derivative. The sulfur atom of the thiol of the thiophosphate group undergoes a nucleophilic reaction with the thiosulfonate group (MeSO ₂ S—) of the compound (3), and the compound (3) and the nucleic acid derivative (A2) are disulfide bonded by de-MeSO ₂ H. To form a phenyldiazirine- added nucleic acid derivative (D2).

Scheme 7 is an example in which the compound (2a) is used as the phenyldiazirine compound. The nucleic acid derivative (A2) is an example having a thiophosphate group at the terminal nucleotide of the nucleic acid derivative. Hydrogen with a compound of the thiol thiophosphate group and Br of (2a) forms a HBr, by de-HBr, compound (2a) with a nucleic acid derivative (A2) and is attached, a phenyl diazirine additional nucleic acid derivative (D3) Generate.

Scheme 8 is an example in which the compound (1) is used as the phenyldiazirine compound. The nucleic acid derivative (A2) is an example having a thiophosphate group at the terminal nucleotide of the nucleic acid derivative. Hydrogen compounds of thiol thiophosphate group (1) transition to the compound (1) and nucleic acid derivative (A2) and is then combined to produce a phenyl diazirine additional nucleic acid derivative (D4).

The phenyldiazirine- added nucleic acid derivative can be a compound represented by the general formula (D). The invention, together with a manufacturing method of the phenyl diazirine additional nucleic acid derivatives also encompasses phenyl diazirine additional nucleic acid derivative itself.

In general formula (D), x and y, and N are the same as defined in general formula (A). R ₄ can be any one of the following general formulas. In the following general formula, n can be an integer of 1 to 6, for example.

Phenyl diazirine additional nucleic acid derivative can be a one having a label. The label can be a known label used for nucleic acid derivatives. Such labels can include, for example, biotin, radioisotopes, and / or fluorescent materials. However, it is not limited to these substances.

[Method for producing phenyldiazirine- added nucleotide derivative (2)]
The method (2) for producing a phenyldiazirine- added nucleotide derivative of the present invention comprises a nucleotide derivative represented by the following general formula (E) or (F) and a thiol group in the compound represented by the general formula (E) or (F) comprising reacting a phenyl diazirine compound having a reactive group as.

  In the general formula (E), R is any base selected from the group consisting of adenine, guanine, cytosine, uracil, thymine and derivatives thereof. Examples of the base derivative include, for example, 5-methylcytosine, N6-methyl It can be, but is not limited to, adenine, 5-hydroxymethylcytosine, inosine and the like. n is 0, 1 or 2.

In the general formula (F), R ₁ and R ₂ are independently selected from the group consisting of nicotinamide adenine nucleotide and its phosphooxide, and flavin mononucleotide, sugar phosphate, sugar nucleotide, coenzyme A, and phospholipid. It is.

  Examples of the phosphoric acid oxide of nicotinamide adenine nucleotide include nicotinamide adenine dinucleotide phosphate. Examples of flavin nucleotides include flavin adenine dinucleotide. Examples of the sugar phosphate include glucose 1-phosphate, glucose 6-phosphate, fructose 1,6-bisphosphate, 5-phosphoribosyl 1-diphosphate, and the like. Examples of sugar nucleotides include ADP-sugar, CDP-sugar, GDP-sugar, UDP-sugar, and TDP-sugar. These sugar moieties include various pentoses, hexoses, uronic acids, deoxy sugars, amino sugars, and branched sugars. , Aminouronic acid, ketose, sulfate sugar, oligosaccharide and the like. Examples of coenzyme A include acetyl coenzyme A. Examples of the phospholipid include glycerophospholipid, phosphatidylcholine (lecithin), phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol (cardiolipin), sphingophospholipid and the like.

The phenyldiazirine compound is a compound represented by the general formula (C), and this compound is the same as the substance described in the production method (1) of the present invention.

[Protein analysis method by electrical mobility shift assay]
The present invention includes a method for analyzing proteins by an electrical mobility shift assay. In this analytical method, using a phenyl diazirine additional nucleic acid derivatives or nucleotide derivatives prepared by the method (2), the phenyl diazirine additional nucleic acid derivatives and the present invention prepared by the method (1) of the present invention.

The analysis method includes the following steps.
(A) mixing the protein that is the subject and phenyl diazirine additional nucleic acid derivatives or nucleotide derivatives under conditions capable of interacting.
And irradiating light to (b) the resulting mixture, reacting the diazirine groups and proteins contained in the phenyl diazirines additional nucleic acid derivatives or nucleotide derivatives, conjugates of the phenyl diazirine additional nucleic acid derivatives or nucleotide derivatives and proteins Form.
(C) The obtained conjugate is subjected to electrophoresis.

Step (a)
The nucleic acid derivative or nucleotide derivative and the protein as the analyte are mixed under conditions that allow interaction. The nucleic acid derivative is prepared so as to have an arbitrary sequence considering the sequence of the protein as the analyte. The prepared nucleic acid derivative (for example, DNA) is used for interaction with a protein after purification if necessary. A nucleic acid derivative having a tag such as biotin introduced at the 5 ′ end can be used.

  The protein as the analyte can be, for example, various transcription factor proteins typified by p53, or various enzyme groups involved in various transcriptions typified by DNA polymerase. At this time, if a protein that binds to the nucleic acid derivative is present in the protein mixture, the two are bound by their affinity. The conditions that can interact can be, for example, standing for about 1 hour in ice. The nucleic acid derivative or nucleotide derivative can be a single component, but a mixture containing a plurality of types of nucleic acid derivatives or nucleotide derivatives can also be used.

Step (b)
The resulting mixture is irradiated with light to cause the diazirine group contained in the phenyldiazirine- added nucleic acid derivative or nucleotide derivative to react with the protein. The light to be irradiated can be, for example, light around 360 nm. Conditions such as irradiation time vary depending on the intensity of the light source, but can be, for example, about several seconds to 30 minutes on ice. By the light irradiation, a covalent bond is formed between the phenyldiazirine- added nucleic acid derivative and the protein, and the two are connected.

By the light irradiation, the diazirine group of the phenyldiazirine- added nucleic acid derivative or nucleotide derivative reacts with an arbitrary part of the protein to form a conjugate.

Step (c)
In step (c), the resulting conjugate is subjected to electrophoresis. The electrophoresis can be normal gel electrophoresis. Specifically, a sample is placed on top of the slab gel and an appropriate amount of current is passed to flow through the slab gel depending on the charged state of the sample. When passing through the gel network, the mobility varies depending on the molecular weight of the sample, and separation analysis becomes possible.

  Furthermore, electrophoresis can be performed under denaturing conditions, and it is preferable to perform under denaturing conditions from the viewpoint of higher separation and higher reproducibility. The denaturing condition means, for example, that the higher-order structure of the protein is broken by a reducing agent or heat treatment, and the surfactant is coexisted to make the charge of the sample uniform.

  The conjugate separated by electrophoresis can be detected using a tag introduced into a nucleic acid derivative. Detection of the conjugate can be appropriately performed depending on the type of tag.

[Protein preparation method]
The present invention encompasses a method for preparing a protein. In this preparation method, phenyl diazirine additional nucleic acid derivatives prepared by the process of the present invention (1), the nucleotide derivative prepared by the method (2) phenyl diazirine additional nucleic acid derivatives of the present invention or used.

The protein preparation method includes the following steps.
(D) Mixing under conditions that allow the phenyldiazirine- added nucleic acid derivative or nucleotide derivative to interact with the protein of interest .
By irradiating light to the resulting mixture (e), by reacting a diazirine group and proteins contained in the phenyl diazirines additional nucleic acid derivatives or nucleotide derivatives, conjugates of the phenyl diazirine additional nucleic acid derivatives or nucleotide derivatives and proteins Form.
(F) separating the conjugate.
(G) The protein is recovered by treating the separated conjugate with an alkaline solution to dissociate the conjugate.

  Steps (d) and (e) can be performed in the same manner as steps (a) and (b).

Step (f)
For selection of the conjugate to be separated, for example, a conjugate of a nucleic acid derivative in which a tag such as biotin is introduced at the 5 ′ end and a protein can be separated by an affinity separation method for the tag. In addition, for example, when using a nucleic acid derivative in which a tag such as biotin is introduced at the 5 ′ end, for example, the conjugate is captured with beads immobilized with avidin as a complex with a protein, Can be implemented.

Step (g)
The separated conjugate is treated with an alkaline solution to dissociate the conjugate. The treatment of the conjugate with an alkaline solution can be performed, for example, as follows. The cleavage between the phosphorus and sulfur atoms is due to a nucleophile. The separated complex is transferred into a solution adjusted to pH 10.5 with 50 mM phosphoric acid and sodium hydroxide, and allowed to stand at 37 ° C. overnight. This cleavage reaction can be referred to Gish, G., Eckstein, F. Science (1988) 240, 1520-1522. The excised binding protein is present in the solution. The state of dissociation of the conjugate is shown by the chemical formula in the following scheme 9.

  Hereinafter, the present invention will be described in more detail with reference to examples.

Reference example 1
Method for synthesizing compound of formula (1)

N- [4- [3- (trifluoromethyl) -3H-diazirin-3-yl] benzyl] maleimide (1)
4- [3- (Trifluoromethyl) -3H-diazirin-3-yl] benzyl alcohol (13) (750 mg, 3.45 mmol) is dissolved in 28 mL of THF, triphenylphosphine (1.05 g, 3.9 mmol), maleimide (389 mg, 3.97 mmol) was added and cooled to 0 ° C. Diisoproylazodicarboxylate (789 mg, 4.35 mmol) was slowly added with stirring under a nitrogen stream. When the addition was completed, it became a yellow liquid. After 1 hour at 0 ° C., the reaction mixture is distilled off under reduced pressure and 30 mL of Hezane: Ether = 1: 1 is added. The precipitated reaction product, Triphenylphosphine oxide, was removed by filtration. The solvent was distilled off under reduced pressure, and the residue was purified by silica gel chromatography (Hexane: AcOEt = 4: 1) to obtain a colorless solid substance.
Yield 539 mg (68%)
¹ H-NMR (CDCl ₃ )
: 7.37 (d, 2H, J = 7.3 Hz) 7.19 (d, 2H, J = 7.3 Hz) 6.72 (s, 2H) 4.67 (s, 2H).

Reference Example 2-1

4- [3- (Trifluoromethyl) -3H-diazirin-3-yl] benzaldehyde (12)
3- (Phenyl) -3- (trifluoromethyl) -3H-diazirine (11) (1.86 g, 0.01 mol) was dissolved in Cl ₂ CHOCH ₃ (3.45 g, 0.03 mol) at 0 ° C. A yellow solid obtained from TfOH (1.77 mL, 0.02 mol) and TiCl ₄ (2.85 g, 0.015 mol) was slowly added with stirring at 0 ° C. under an argon stream. When the generation of hydrogen chloride subsided, an orange liquid was formed. After leaving at room temperature for 1 hour, 500 mL of hexane was added to the reaction mixture, crushed ice was slowly added with stirring under ice to decompose excess reagent, and then solid sodium carbonate was slowly added to neutralize. The obtained hexane layer was separated, washed with saturated brine, and dried over magnesium sulfate. After removing the desiccant by filtration, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (Hexane: CH ₂ Cl ₂ = 2: 1) to obtain a yellow oily substance (12).

Yield 1.71 g (80%)
¹ H-NMR (CDCl ₃ )
σ: 10.04 (s, 1H), 7.91 (d, 2H, J = 8.3 Hz) 7.35 (d, 2H, J = 8.3 Hz).

Reference Example 2-2

  As described above, in the above scheme, (13) is the same compound as the compound (7), (14) is a compound in which Hal is Br in the compound (2), and (15) is In the above (10), R and R ′ are hydrogen atoms (H).

4- [3- (Trifluoromethyl) -3H-diazirin-3-yl] benzyl alcohol (13)
Compound (12) (3.3 g, 15.4 mmol) was dissolved in EtOH 7 mL, and NaBH ₄ (0.626 g, 16.5 mmol) suspended in EtOH 7 mL was added with stirring at 0 ° C. After the addition was completed, the mixture was returned to room temperature and stirred for 4 hours. To this, 1 M HCl was slowly added on ice to decompose excess reagent, followed by extraction with Ether, and the Ether layer was dried over magnesium sulfate. After removing the desiccant by filtration, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (Hexane: Ether = 1: 1) to obtain a pale yellow oily substance (13).

Yield 3.21 g (96%)
¹ H-NMR (CDCl ₃ )
σ: 7.39 (d, 2H, J = 8.5 Hz), 7.19 (d, 2H, J = 8.5 Hz), 4.71 (s, 2H), 1.87 (bs, 1H)

Reference Example 2-3
3- [4- (Bromomethyl) phenyl] -3- (trifluoromethyl) -3H-diazirine (14)
Compound (13) (344.0 mg, 1.59 mmol) was dissolved in 2 mL of CH ₂ Cl ₂ and CBr ₄ (658.2 mg, 1.985 mmol) was added. Cooled to _{0 ℃ Ph 3 P (474 mg} , 1.807 mmol) was added slowly. After returning to room temperature and stirring for 1 hour, the residue was purified by silica gel column chromatography (Hexane) to obtain a colorless oily substance (14).

Yield 401.8mg (90%)
¹ H-NMR (CDCl ₃ )
σ: 7.42 (d, 2H, J = 8.5 Hz), 7.17 (d, 2H, J = 8.5 Hz), 4.46 (s, 2H).

Reference Example 2-4
L-4 '-[3- (Trifluoromethyl) -3H-diazirin-3-yl] phenylalanine (15)
tert-Butylglycinate benzophenone imine (1.136 g, 3.839 mmol) and the asymmetric catalyst O (9) -Allyl-N-9-anthracenylmethylcinchonidium bromide ^[9] (0.2127 g, 0.351 mmol, 0.1 eq) in CH ₂ Cl ₂ 9 Dissolved in mL. Compound (14) (1.419 g, 5.085 mmol, 1.5 eq.) Was added, and the mixture was stirred at room temperature under an argon gas atmosphere. While cooling the reaction mixture to -78 ° C, 2-tert-Butylimino-2-diethylamino-1,3-dimethylperhirdo-1,3,2-diazaphosphorine (1.41 g, 5.139 mmol, Aldrich) was added dropwise in a few seconds, The reaction was carried out at -78 ° C for 7 hours. After returning to room temperature and distillation under reduced pressure, the residue was diluted with Ether and washed twice with distilled water and once with saturated saline. The oil layer was dried with magnesium sulfate. After removing the desiccant by filtration, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (Hexane: EtOAc = 7: 1) to obtain a yellow oily substance.

Yield 1.89 g (95%)
¹ H-NMR (CDCl ₃ )
σ: 7.57 (d, 2H, J = 6.9 Hz), 7.39-7.27 (m, 6H), 7.06 (q, 4H, J = 8.4 Hz)
6.59 (d, 2H, J = 6.9 Hz), 4.09 (q, 1H, J = 8.6, 4.6 Hz), 3.26-3.11 (m, 2H)
1.44 (s, 9H).

The obtained yellow oily substance (75 mg, 0.152 mmol) was dissolved in 1 mL of TFA at 0 ° C. and stirred at room temperature for 2 hours. After confirming the completion of the reaction with (TLC, Hexane: AcOEt = 7: 1), TFA was distilled off under reduced pressure, and the residue was diluted with CH ₂ Cl ₂ and extracted three times with water. The aqueous layer was lyophilized to give a white solid (15).

Yield (as TFA salt) 53.4 mg (91%)
¹ H-NMR (CD ₃ OD)
σ: 7.46 (d, 2H, J = 7.9 Hz), 7.27 (d, 2H, J = 7.9 Hz), 3.84 (dd, 1H, J = 8.2, 4.5 Hz), 3.38 (m, 1H), 3.10 (d , 1H, J = 4.5, 8.2 Hz).
Optical purity (ee)
(5) was induced to Fmoc form, and its optical purity was confirmed by HPLC. After dissolving an appropriate amount in MeOH, elute it with 0.01 M Ammonium Acetate / MeOH at a flow rate of 1 mL / min using a chiral column (Sumichiral OA-3300 2.5 μm 4.6 mmφ × 25 cm), and absorb UV light derived from 360 nm diazirine. Detected.
ee = 98%
[α] _D + 22.4 ° (c = 0.52, EtOH)

Example 1
As a nucleic acid derivative, a compound (AGsCT) in which a phosphoric acid between guanine and cytosine of 4-mer DNA having adenine, guanine, cytosine, and thymine sequences represented by compound (A3) in the following scheme 10 is used as a thioester It was. This compound was synthesized by a known method described in the following literature. Zon, G .; Geiser, TG (1991) Phosphorothioate oligonucleotides: chemistry, purification, analysis, scale-up and futuredirections.Anticancer Drug Des. 6: 539-568. And Caruthers, MH; Beaton, G .; Wu, JV; Wiesler, W. (1992) Chemical synthesis of deoxyoligonucleotides and deoxyoligonucleotide analogs.Methods Enzymol. 211: 3-20.

  Compound (2a) in Scheme 3 with a molar ratio of 100 times and diisopropylethylamine with a molar ratio of 200 times were mixed with DNA containing 0.05 mM AGsCT (A3) containing sulfur atoms. The reaction was carried out under conditions of room temperature, 37 ° C., and 56 ° C. The reaction time was 15 minutes to 24 hours, and the purification rate of the product (D5) was followed by high performance liquid chromatography. The results are shown in Figure 1.

  The chart shown in FIG. 1 is an analysis example by high performance liquid chromatography when the reaction is performed at 37 ° C. Chart 1 is before the reaction, and charts 2, 3, 4, and 5 are those after 1, 2, 4, and 24 hours after the reaction, respectively. The number shown at the top of the peak indicates the retention time, and the peak near 6.5 minutes is the compound (A3) in Scheme 10 of the raw material, and the peak near 7.5 minutes is the compound (D5) in Scheme 10 of the product.

As a result of fractionating peak portions corresponding to the compound (A3) and the compound (D5) and performing mass spectrometry, measurement of 1191 (M + H ⁺ ) in the compound (A3) and 1388 (M + H ⁺ ) in the compound (D5) The result was obtained. These agree well with the theoretical mass values of 1191.22 (M + H ⁺ ) and 1388.26 (M + H ⁺ ), which are the theoretical mass values of compound (A3) and compound (D5), indicating that compound (D5) can be produced from compound (A3).

  Graphs (1), (2), and (3) shown in FIG. 2 show the reaction time and the yield of the product compound (D5) when the reaction was performed at 20 ° C., 37 ° C., and 56 ° C., respectively. Yes. The yield is good at the reaction temperature of 37 ° C. in the graph (2). The maximum yield is obtained by the reaction for about 2 hours.

  The reaction yield was measured by changing the concentration of the compound (A3) in Scheme 10 as a raw material. The results are shown in Figure 3.

  The reaction temperature was fixed at 37 ° C., and the reaction yields when the concentration of the compound (1) in Scheme 3 as the raw material was 0.023, 0.045, 0.182, 0.455 mM were shown in graphs (1), (2), (3), respectively. (4). The reaction is faster when the raw material is at a high concentration, but there is no change around 0.2 mM. Although the preferable raw material concentration is 0.2 mM, it is considered that a similar yield can be obtained by extending the reaction time even if it is about 0.1 mM.

Example 2

A 21-mer RNA (A2) thiophosphorylated at the 5 ′ end and a phenyldiazirine compound represented by formula (1) were prepared in dityl sulfoxide so that the final concentrations were 0.025 mM and 20 mM, respectively. Further, diisopropylethylamine was mixed so that the final concentration was 20 mM, and the mixture was allowed to stand at 37 ° C. for 2 hours. The sample after the reaction was analyzed by high performance liquid chromatography. The results are shown in FIG.

  The chart (1) shown in FIG. 4 is an analysis result by high performance liquid chromatography of the raw material (A2). The peak corresponding to (a) in the chart represents 21-mer RNA (A2) obtained by thiophosphorylating the 5 ′ end of the raw material. The chart (2) is a similar analysis of the product (D4). It was confirmed that the peak (b) in the chart was a newly appearing peak and was (D4) as a result of mass spectrometry.

Example 3

  An example will be shown in which a photoreactive DNA produced by the method of the present invention is used to simultaneously analyze a large number of proteins that bind to the DNA sequence. Biotin-TGTATGsCAAATAAGG (SEQ ID NO: 1) (A1) in which the photoreactive group and 5 ′ end of the compound represented by the formula (2a) are biotinylated and one of the nucleic acids in the DNA sequence is a thiophosphate ester, It was dissolved in dimethyl sulfoxide to a concentration of 0.2 mM and 10 mM. Diisopropylethylamine was mixed to a final concentration of 10 mM to synthesize a product represented by the formula (D1). The target product was purified by high performance liquid chromatography, and the product was confirmed by mass spectrometry. Compound A1 was synthesized by a known method described in the following literature. Zon, G .; Geiser, TG (1991) Phosphorothioate oligonucleotides: chemistry, purification, analysis, scale-up and futuredirections.Anticancer Drug Des. 6: 539-568. And Caruthers, MH; Beaton, G .; Wu, JV; Wiesler, W. (1992) Chemical synthesis of deoxyoligonucleotides and deoxyoligonucleotide analogs.Methods Enzymol. 211: 3-20.

  0.1 μg of the product represented by the chemical formula (D1), 25 μg of HeLa cell nuclear extract, 25 mM HEPES-Na, 50 mM NaCl, 5 mM DTT, 1 mM EDTA, 0.05% Triton X-100, 10% glycerol, 2 μg poly (dI · dC), dissolved in 20 μL of a mixed solution of 100 μg / mL BSA, and allowed to stand in ice for 1 hour. When binding inhibition was performed, 10 to 100-fold molar amount of the competitive DNA to be inhibited was allowed to coexist.

The tube containing the sample was floated on ice water and irradiated with 360 nm light of 30 W / m ² on ice for 1 to 5 minutes. Immediately after the irradiation, 5 μL of electrophoresis denaturing solution was added, and the mixture was allowed to stand at room temperature for 30 minutes or more. A schematic diagram of this operation is shown in FIG.

  Samples containing covalently bound DNA and binding protein were separated by denaturing electrophoresis. The separation gel at this time was made of 10% polyacrylamide. After electrophoresis, the gel was removed, the separated gel was immersed in a fixing solution, and transferred from the gel to the membrane with a semi-dry blotting apparatus. The membrane was blocked with 1.0% (w / v) casein / PBS for 1 hour at room temperature, and then 0.1% (v / v) Strept-avidin HRP / PBS-T was added and shaken at room temperature for 30 minutes. After washing the membrane several times, biotin was detected with a chemiluminescent reagent.

  Biotin is originally bound to photoreactive DNA. When a single photoreactive DNA is analyzed by electrophoresis using a 10% polyacrylamide gel, the molecular weight is as small as about 5000, so that it flows out of the gel. Biotin detected by chemiluminescence in this electrophoresis gel is considered to be photoreactive DNA that forms a complex with a high-molecular substance such as protein.

  The lanes in the analysis example shown in FIG. 6 will be described. Lane “M” shows molecular weight markers, corresponding to molecular weights of 67000, 45000, 31000, 20000 from the top of the gel. Lane “1” is a control experiment in which no nuclear extract was added and the experiment was carried out using only photoreactive DNA. Lane “2” is an experiment in which the nuclear extract and photoreactive DNA were added and irradiated with light as described above. Lanes “3” and “4” are experiments using competitive inhibition DNA, and Lane “3” is a 25-fold molar amount of DNA having a sequence equivalent to photoreactive DNA. “4” is obtained by adding a 25-fold molar amount of DNA obtained by mutating a part of the DNA sequence.

  Comparison of lanes “1” and “2” shows that many binding proteins could be analyzed simultaneously. Inhibition experiments in lanes “3” and “4” indicate that these complex formations are specific to the DNA sequence.

Example 4
[Example of cutting method]
An example in which a dimerin introduced into a 4-mer DNA fragment of AGsCT was cleaved as a sample by the same reaction as the scheme shown in Example 3 is shown. The photoreactive DNA was dissolved in a solution adjusted to pH 10.5 with sodium hydroxide so that the photoreactive DNA was 10 μM. Samples that were allowed to stand at 37 ° C. for 2 hours were analyzed by high performance liquid chromatography. The results are shown in FIG.

  The upper chart (1) is obtained by analyzing the photoreactive AGsCT before the reaction using a high performance liquid chromatography as a sample, and the peak (a) in the chart corresponds to it. Chart (2) is obtained by analyzing the sample after reacting for 2 hours by the above method. The peak corresponding to (a) disappears and the peak (b) newly appears. Chart (3) is obtained by analyzing AGsCT without diazirine as a sample, and the peak of (c) corresponds to it. Since the retention times of the peak of (b) and the peak of (c) are almost the same, the peak of (b) is expected to be AGsCT in which diazirine is cleaved. As a result of fractionating this (b) peak and analyzing it by mass spectrometry, it was shown that it was AGsCT without diazirine.

Example 5
[Example of cutting]
Photoreactive DNA was produced from Biotin-TGTATGsCAAATAAGG (SEQ ID NO: 1) having biotin at the 5 ′ end in the same reaction as the scheme shown in Example 3. A schematic diagram of the example is shown in FIG.

  The photoreactive DNA is allowed to stand in a polypropylene tube (FIG. 8 (1)) and fixed to the tube surface by light irradiation (FIG. 8 (2)). Unimmobilized DNA is removed by washing (FIG. 8 (3)), and the presence or absence of biotin is confirmed by streptavidin-HRP enzyme by chemiluminescence (FIG. 8 (4)). A cutting process was performed after the operation shown in FIG. 8 (3), and it was confirmed whether biotinylated DNA was removed from the tube surface by cutting.

Detailed experimental procedures are as follows.
1. Dry 50 μL of 100 μM photoreactive DNA (in water) in a PCR tube.
2. Fix DNA to the tube by photoreaction (360 nm, 5 min).
Wash with 100% 3.1% Tween, 50 mM Tris-HCl (pH 7.4), 300 mM NaCl. (3 times)
Wash with 100 μL of 4.50 mM Tris-HCl (pH 7.4), 300 mM NaCl. (Tween removal)
5. Cut PS under cutting conditions <Condition 1> Add 100 μL of a solution adjusted to pH 10.5 with 50 mM phosphate buffer + NaOH, and leave at 37 ° C. for 2 hours.
<Condition 2> Add 100 μL of 50 mM Acetate-Na buffer (pH 3.0) 100 mM NaCl and leave at 37 ° C. overnight (about 15 hours).
Add 100 μL of 6.1% casein PBS and leave at 37 ° C for 1 hour. (blocking)
7. Wash with 100 μL of 0.1% Tween, 50 mM Tris-HCl (pH 7.4), 300 mM NaCl. (3 times)
8. Add streptavidine-HRP in 50 mM Tris-HCl (pH 7.4), 300 μL of 300 mM NaCl, and leave at 37 ° C for 1 hour.
9. Wash with 100 μL of 0.1% Tween, 50 mM Tris-HCl (pH 7.4), 300 mM NaCl.
10. ECL measurement. The results are shown in FIG.

  One of the chemiluminescence detection examples in FIG. 9 is one in which no cleavage treatment is performed, and shows that biotinylated DNA is immobilized on the tube surface. In the detection example 2, no photoreaction is performed and no immobilization treatment is performed. Detection Example 3 is an example of cleavage treatment, which was left overnight in a solution of 50 mM phosphate buffer adjusted to pH 10.5 with sodium hydroxide. This shows that biotinylated DNA has been removed from the tube surface by the cutting treatment.

Example 6
[Example of separation of binding protein]
An example in which a protein that binds to the DNA sequence is separated using photoreactive DNA will be described. In the same scheme as in Example 3, photoreactive DNA was synthesized from Biotin-TGTATGsCAAATAAGG (SEQ ID NO: 1) in which the 5 ′ end was biotinylated, and the target product was purified by high performance liquid chromatography. 10 μM of photoreactive DNA and 7.8 μg of protein that binds to this DNA sequence were combined with 25 mM HEPES-Na, 50 mM NaCl, 5 mM DTT, 1 mM EDTA, 0.05% Triton X-100, 10% glycerol, 2 μg poly (dI · dC), dissolved in 20 μL of a mixed solution of 100 μg / mL BSA, and allowed to stand in ice for 1 hour.

The tube containing the sample was floated on ice water and irradiated with light of 360 nm with a light amount of 30 W / m ² on ice for 5 minutes. A surfactant was added to a final concentration of 1% immediately after irradiation. Avidin-bound beads were added and stirred for 1 hour. After washing, the cleavage reaction was carried out in a solution of 50 mM phosphate buffer adjusted to pH 10.5 with sodium hydroxide. The supernatant was collected, separated by electrophoresis, and the protein was stained with silver stain. The results are shown in FIG.

  The lanes in the electropherogram of FIG. 10 will be described. Lane “M” shows molecular weight markers, corresponding to molecular weights of 97000, 67000, 45000, 31000, 20000 from the top of the gel. Lane “1” uses the supernatant that has been cleaved as described above as a sample, lane “2” uses the sample that has been cleaved without adding POU protein as a sample, and lane “3” uses POU protein as a sample. It was. From lane “3”, “A” and “B” in the electropherogram are considered to be POU protein bands. These two bands can also be seen in lane “1”. It shows that cleavage occurred between the POU protein and DNA, and the protein captured by the photoreaction could be recovered.

Example 7
[Introduction of diazirine into GTP-γ-S, ADP-β-S]

Diazirine was introduced into the phosphorothioates of GTP-γ-S and ADP-β-S shown below.

<Operation>
-Preparation of photoreactive GTP-γ-S The final concentration of the sample is 1 mM GTP-γ-S, 100 mM phosphate buffer (pH 7.0), 50 mM 4- [3H- [3-trifluoromethyl] diazine-3- Prepare yl] benzyl maleimide and leave at 37 ° C for 20 hours. After the reaction, analysis and isolation were performed by HPLC, and molecular weight was measured by MASS.

-Preparation of photoreactive ADP-β-S The final concentration of the sample is 1 mM ADP-β-S, 100 mM phosphate buffer (pH 7.0), 50 mM 4- [3H- [3-trifluoromethyl] diazine-3- yl] benzyl maleimide was prepared and placed at 37 ° C. for 18 hours. After the reaction, analysis and isolation were performed by HPLC, and molecular weight was measured by MASS. The results are shown in FIG.

From the HPLC results, a new product peak was confirmed at 14.2 min for GTPγ-S and 14.0 min for ADP-β-S in the diazirine introduction reaction. This peak was collected and the molecular weight was measured by MASS.

  The results of molecular weight measurement confirmed the introduction of diazirine into GTP-γ-S and ADP-β-S. Each yield was 94.1% for GTP-γ-S and 87% for ADP-β-S. Similar to the introduction of diazirine into GTP-γ-S and ADP-β-S, photoreactive groups can be introduced into various phosphorothioates.

Example 8
Photoaffinity label of photoreactive GTP and H-Ras

1 mL of a sample containing 40 ng H-Ras, 10 mM diazirine-GTP, 10 mM MgCl ₂ and 50 mM Tris-HCl (pH 7.4) was prepared and incubated on ice for 30 minutes. Subsequently, 360 nm UV (15 w x 2) was irradiated on ice for 10 minutes *. Add 40 mL of 1 mM N-ethylmaleimide (NEM), 1 mM tri-n-butylphosphine, 0.5 M phosphate buffer (pH 7.0), 10% ethanol composition to the sample, and incubate at 20 ° C under argon for 20 hours did. NEM was removed from the sample after the reaction using a Sephadex G25 column, and the PS in the photoreactive GTP was cleaved by hydrolysis by incubating with a 0.03% aqueous ammonia solution at 37 ° C. for 2 hours. After the reaction, ammonia was removed by aeration of nitrogen, 40 mL of 1 mM biotinmaleimide 0.5 M phosphate buffer (pH 7.0) and 10% ethanol composition was added, and the mixture was incubated at 20 ° C. for 20 hours under argon. After the reaction, biotinmaleimide was removed from the sample using a Sephadex G25 column and lyophilized. Samples were subjected to SDS-PAGE (13% polyacrylamide gel), electroblotted onto a PVDF membrane, and then detected by chemiluminescence using streptavidin-HRP peroxidase. The results are shown in FIG. The reaction scheme is shown below.

Example 9
Isolation and detection of optical label Ras using GTP tag
1 mL of a sample containing 40 ng H-Ras, 10 mM diazirine-GTP, 10 mM MgCl ₂ , 50 mM Tris-HCl (pH 7.4) was prepared and incubated on ice for 30 minutes †. Subsequently, 360 nm UV (15 w x 2) was irradiated on ice for 10 minutes *. After the photoreaction, 40 mL of an aqueous solution containing 1 mM biotin-O succinimide, 50 mM Tris-HCl (pH 7.4) and 10% acetonitrile was added and reacted at 37 ° C. for 2 hours. Thereafter, 4 mL of 10% SDS was added and the protein was denatured by heating at 95 ° C for 5 minutes. Then added 0.2 M aqueous acetic acid solution an equal volume (45 mL), was placed on Fe ³⁺ -IMAC column (50mL min) ^$. After washing with 1 mL of 0.1 M acetic acid aqueous solution and 0.1% acetic acid / 60% acetonitrile aqueous solution, the eluate was lyophilized by elution with 500 mL of 0.1 M ammonia aqueous solution. The sample was dissolved in 10 mL water, and the entire amount was subjected to SDS-PAGE (13% polyacrylamide gel). After electroblotting on a PVDF membrane, detection was performed by chemiluminescence using streptavidin-HRP peroxidase. The results are shown in FIG. The reaction scheme is shown in FIG.

† Results 3-5 added GTP for inhibition at this point.
* Result 2 does not perform this light irradiation.
^$ Fe ³⁺ -IMAC shows a phosphate group and a chelate. In this case, only the photolabeled GTP bound to the protein will chelate with the column.

  The present invention is useful in a wide range of fields involving nucleic acids and proteins.

An example of analysis by high performance liquid chromatography is shown. The relationship between reaction time and yield is shown. The relationship between reaction concentration and yield is shown. An example of analysis by high performance liquid chromatography is shown. Schematic diagram in which a covalently bound DNA and a binding protein are formed. An example of complex analysis detected by chemiluminescence is shown. An example of analysis by high performance liquid chromatography is shown. FIG. 6 is a schematic diagram of Example 6. An example of chemiluminescence detection is shown. Electrophoresis image of the collected supernatant. Analysis of introduction reaction of diazirine into GTP-γ-S by HPLC. Analysis of introduction reaction of diazirine into ADP-β-S by HPLC. The chemiluminescence detection result of the optical label Ras by optical probe specific biotin addition in Example 8. 1: With optical label 2: Without optical label (no operation * light irradiation) FIG. 6 shows the results of isolation and detection of optically labeled Ras using GTP-tag in Example 9. FIG. 1: With optical label 2: Without optical label 3: Inhibition by equivalent GTP 4: Inhibition by 10 equivalent GTP 5: Inhibition by 100 equivalent GTP The reaction scheme in Example 9.

Claims (13)

Comprises reacting a phenyldiazirine compound having a thiol group of the nucleic acid derived body and thiophosphate group is a compound represented by the general formula (A) The reactive groups,
Ri compounds der said phenyldiazirine compound represented by the general formula (C), and
A method for producing a phenyldiazirine-added nucleic acid derivative, wherein the phenyldiazirine-added nucleic acid derivative is a compound represented by the general formula ( D ) .
In general formula (C), R ₅ is
R ₆ is a halogen atom ;
R ₇ is an alkylsulfonyl group having 1 to 6 carbon atoms,
R ₈ is H ;
n is an integer of 1-6.
In the general formula ( A ) , x and y are each independently an integer of 0 to 100, and N is a group represented by the following general formula (B).
In general formula (B), R ₁ is an OH group or an adjacent nucleotide, R ₂ is a hydrogen or OH group, R ₃ is an OH group or an adjacent nucleotide, and B is adenine, guanine, cytosine, uracil. , Thymine, 5-methylcytosine, N6 -methyladenine, 5-hydroxymethylcytosine, and inosine, and X is S (sulfur) or O (oxygen).
In general formula (D), x and y and N are the same as defined in general formula (A), and R ₄ is
And n is an integer of 1-6. The method according to claim 1, wherein the phenyldiazirine compound represented by the general formula ( C ) is a compound represented by the following formulas (1) to ( 3 ).
The process according to any one of claims 1-2 as nucleic acid derivatives and / or phenyldiazirine additional nucleic acid derivatives are those having a label. Label, biotin method according to claim 3 radioisotope, or is a fluorescent substance. A phenyldiazirine-added nucleic acid derivative represented by the general formula (D).
In general formula (D), R ₄ is
N is an integer of 1 to 6, x and y are independently an integer of 0 to 100, and N is a group represented by the following general formula (B).
In general formula (B), R ₁ is an OH group or an adjacent nucleotide, R ₂ is a hydrogen or OH group, R ₃ is an OH group or an adjacent nucleotide, and B is adenine, guanine, cytosine, uracil. , thymine, 5-methylcytosine, N6 - methyl adenine, 5-hydroxymethyl cytosine, and salts group Ru is selected from the group consisting of inosine, X is S (sulfur) or O (oxygen). The derivative according to claim 5 , wherein the phenyldiazirine-added nucleic acid derivative has a label. Label, derivative according to claim 6 biotin, radioisotope, or is a fluorescent substance. Reacting a nucleotide derivative represented by the following general formula (E) or (F) with a phenyldiazirine compound having a reactive group with a thiol group in the compound represented by the general formula (E) or (F) And the phenyldiazirine compound is a compound represented by the general formula (C).
In the general formula (E), R is adenine, guanine, cytosine, uracil, thymine, 5-methylcytosine, N6 - methyl adenine, 5-hydroxymethyl cytosine, and salts group Ru is selected from the group consisting of inosine, n Is 0, 1 or 2, and in general formula (F), R ₁ and R ₂ are independently nicotinamide adenine nucleotide and its phosphate, and flavin mononucleotide, sugar phosphate, sugar nucleotide, coenzyme A, and selected from the group consisting of phospholipids.
In general formula (C), R ₅ is
R ₆ is a halogen atom;
R ₇ is an alkylsulfonyl group having 1 to 6 carbon atoms ,
R ₈ is H;
n is an integer of 1-6. The method according to claim 8 , wherein the phenyldiazirine compound represented by the general formula (C) is a compound represented by the following formulas (1) to ( 3 ).
The phenyldiazirine-added nucleic acid derivative produced by the method according to any one of claims 1 to 4, the phenyldiazirine-added nucleic acid derivative according to any one of claims 5 to 7 , or the claims 8 to 9 The nucleotide derivative produced by the method according to any one of the above and a protein to be analyzed are mixed under conditions that allow interaction,
The obtained mixture is irradiated with light to react the diazirine group contained in the phenyldiazirine-added nucleic acid derivative or nucleotide derivative with the protein, and to combine the phenyldiazirine-added nucleic acid derivative or nucleotide derivative with the protein. Forming and then subjecting the resulting conjugate to electrophoresis,
Protein analysis method by electrical mobility shift assay. The method according to claim 10 , wherein the electrophoresis is performed under denaturing conditions. The phenyldiazirine-added nucleic acid derivative produced by the method according to any one of claims 1 to 4, the phenyldiazirine-added nucleic acid derivative according to any one of claims 5 to 7 , or the claims 8 to 9 The nucleotide derivative produced by the method according to any one of the above and a protein to be analyzed are mixed under conditions that allow interaction,
The obtained mixture is irradiated with light to react the diazirine group contained in the phenyldiazirine-added nucleic acid derivative or nucleotide derivative with the protein, and to combine the phenyldiazirine-added nucleic acid derivative or nucleotide derivative with the protein. Forming,
The resulting conjugate is subjected to electrophoresis,
Separating the conjugate, and then treating the separated conjugate with an alkaline solution to dissociate the conjugate;
Recovering the dissociated protein and / or nucleic acid derivative or nucleotide derivative,
Protein preparation method. The method according to claim 12 , wherein the electrophoresis is performed under denaturing conditions.