WO2006120992A1

WO2006120992A1 - Nucleic acid derivatives having phenyldiaziridine moieties and process for production thereof, nucleotide derivatives having phenyldiaziridine moieties and process for production thereof, method for analysis of protein, and process for preparation of protein

Info

Publication number: WO2006120992A1
Application number: PCT/JP2006/309229
Authority: WO
Inventors: Yasumaru Hatanaka; Yutaka Sadakane; Masaki Kaneda
Original assignee: National University Corporation University Of Toyama
Priority date: 2005-05-09
Filing date: 2006-05-08
Publication date: 2006-11-16
Also published as: JP4122446B2; JPWO2006120992A1

Abstract

[PROBLEMS] To provide photoreactive nucleic acid or nucleotide derivatives, a method for comprehensive analysis of protein with the derivatives, and so on. [MEANS FOR SOLVING PROBLEMS] Nucleic acid derivatives having phenyldiaziridine moieties, as represented by the general formula (D): [Chemical formula 1] (D) a process for the production of the derivatives which comprises reacting a nucleic acid derivative wherein at least one of the phosphate groups is a thiophosphate group with a phenyldiaziridine compound; nucleotide derivatives having phenyldiaziridine moieties; a process for the production thereof which comprises reacting a nucleotide derivative represented by the general formula (E) or (F) with a phenyldiaziridine compound having a group reactive with the thiol group of the derivative represented by the general formula (E) or (F): [Chemical formula 11] (E) (F) and a method for analysis of protein by electrophoresis mobility sift assay with the above nucleic acid or nucleotide derivatives having phenyldiaziridine moieties.

Description

Specification

Phenyldiaziridine-added nucleic acid derivatives and their production methods, phenyldiaziridine-added nucleotide derivatives and their production methods, and protein analysis methods and preparation methods

Technical field

[0001] The present invention relates to a phenyldiaziridine-added nucleic acid derivative and a method for producing the same, and a phenyldiaziridine-added nucleotide derivative and a method for producing the same. Furthermore, the present invention relates to a method for analyzing and preparing a protein using a ferrodiaziridine-added nucleic acid derivative and a ferrodiaziridine-added nucleotide derivative.

Background art

[0002] Nucleic acids are basic substances of life and control many functions of life phenomena. A single nucleic acid and its derivative are responsible for various information transmission and energy transmission in the 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 perform various functions in this way is because there are proteins that bind to them. ("Molecular Biology of Cells 4th Edition" by Bruce Alberts, translated by Keiko Nakamura and Kenichi Matsubara, published by Newton Press (Non-Patent Document 1))

[0003] Protein motif analysis is often used as a method for analyzing proteins that bind to nucleic acids and their derivatives. The sites of the protein that bind to the nucleic acid and its derivatives are somewhat similar to each other, and this property is used to predict whether or not the protein will bind to the nucleic acid and its derivatives.

[0004] Electrical mobility shift assay (hereinafter referred to as EMSA) is often used as a method for analyzing a protein that binds to a nucleic acid complex, DNA, or RNA. 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 the complex is formed with mutual affinity during a certain period of time. Let it form. Then it does not bind to the DNA or RNA-protein complex and DNA or RNA is separated by non-denaturing electrophoresis, and the labeled DNA or RNA is visualized with an appropriate detection method. At this time, the complex generally has a slower mobility during electrophoresis than DNA or RNA alone. ("Molecular Cloning A Laboratory Manual 3rd Edition", Sambrook J, Russell DW, pages 17-13-17-22, Cold Spring Harbor Laboratory Press (Non-patent Document 2))

[0005] The present inventors have so far developed a unique high-speed optical affinity method using a diazirine derivative as a photoreactive group. The photoaffinity method is a technology that uses a photoreaction to irreversibly connect specific ligands and partner proteins. 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. Oorman eds., Chemical Genomics, Marcel Dekker Inc., New York. Ppl99-214 (Non-Patent Document 3), Y. Hatanaka, Y. Sadakane (2002) Photoaffinity labeling in drug discovery and developments: Chemical gateway for entering proteom ic frontier. Curr. Top. Med. Chem. 2, 271- 288 4), Yasumaru Hatanaka Structural biological organic chemistry: Probing protein functional structure by photo-affair label, Journal of Organic Synthesis, 56 (7), 581-590, 1998 (non-patent document 5), M Kaneda, Y. Sadakane, Y. Η atanaka, (2003) A Novel Approach for Aftinity- Based Screening of Target Specific Li gands: Application of Photoreactive D—Glyceraldehyde— 3— phosphate Dehydrogenase. Bioconjugate Chem. 14, 849-852. (Non-Patent Document 6), M. Hashimoto, J. Yang, Y. Hat anaka, Y. Sadakane, K. Nakagomi, GD Holman (2002) Improvement in the propert ies of 3—phenyl— «3— trifluoromethyldiazirine based photoreactive bis— glucose probes for GLUT4 following substitution on the phenyl ring. Chem. Pharm. Bull. 50, 1004—10 06 (Patent Document 7), Hatanaka, Y., Kempin, U ., Park, JJ. One-step synthesis of biotinyl photoprobes from unprotected carbohydrates. J. Org. Chem. 65, 5639—5643, 20 00 (Non-Patent Document 8), Hatanaka, Y., Hashimoto, H., Kanaoka , Y. A rapid and efficient method for identifying photoaffinity biotinylated sites within proteins.J. Am. Chem. Soc. 120, 453-454, 1998 (Non-patent document 9), JP 2000-319262 (Patent document 1) )

[0006] The following problems have not been solved in analyzing, separating and purifying nucleic acids, their derivatives and their complexes. It is a solution.

(1) The method of analyzing proteins to which nucleic acids and their derivatives bind from homologous motifs cannot be used as a method of searching for new binding proteins whose amino acid sequences are unknown by direct confirmation methods. ,.

(2) EMSA, which is frequently used for analysis of DNA or RNA binding proteins, is an analysis method that only confirms the presence or absence of binding proteins with very poor resolution due to the limitations of non-denaturing electrophoresis. For example, even when there are multiple binding proteins in the analysis system, EMSA cannot know the number of proteins for which only information on the presence or absence of binding proteins can be obtained. It is well known that multiple proteins form higher-order complexes and bind to DNA or RNA, and the limitations of EMSA have been pointed out as analysis methods.

(3) A general method for separating and purifying a protein that binds to a nucleic acid, its derivative, and its complex from a protein mixture has not yet been established.

[0007] Therefore, 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 covers the protein using the produced derivative. It is an object of the present invention to provide a general analysis method and a binding protein isolation method (protein production method).

Disclosure of the invention

[0008] The present invention for solving the above problems is as follows.

[1]

Including reacting a nucleic acid derivative (wherein at least one of the nucleic acid phosphate groups is a thiophosphate group) with a thiol group of the thiophosphate group and a phenyldiaziridine hydrate compound having a reactive group , A method for producing a nucleic acid derivative added with ferrule-aziridine.

[2]

The production method according to [1], wherein the nucleic acid derivative is a compound represented by the general formula (A).

[Chemical 1]

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).

[Chemical 2]

B

In the general formula (B), R is an OH group or an adjacent nucleotide, and R is hydrogen or OH

1 2 group, R is OH group or adjacent nucleotide, B is adenine, guanine,

Three

Any base selected from the group consisting of tosine, uracil, thymine and derivatives thereof, X is S (sulfur) or o (oxygen).

[3]

The production method according to [1] or [2], wherein the phenyldiaziridine complex is a compound represented by the general formula (C).

[Chemical 3]

In general formula (C), R is

Five

R is a halogen atom or a sulfonate residue,

6

R is an alkylsulfo group having 1 to 6 carbon atoms or an alkylthio group having 1 to 6 carbon atoms.

7

Yes,

R is H or an alkyl group having 1 to 6 carbon atoms,

8

n is an integer of 1-6.

[Four]

The production method according to any one of [1] to [3], wherein the phenyldiaziridine-added nucleic acid derivative is a compound represented by the general formula (D).

[Chemical 5]

(D)

In general formula (D), x and y, and N are the same as defined in general formula (A), and R is

Four

[Chemical 6]

And n is an integer of 1-6.

[Five]

The method according to [3], wherein the phenyldiaziridine complex is a compound represented by the following formulas (1) to (10).

In formulas (4), (5), (6), and (9), R is hydrogen), an unsubstituted or substituted alkyl group having 1 to 6 carbon atoms, and in formula (10), R is Hydrogen), an unsubstituted or substituted alkyl group having 1 to 6 carbon atoms, and R ′ is H, an unsubstituted or substituted alkyl carboalkyl, alkyl carboloxy or aryl carborooxy group having 2 to 6 carbon atoms. In the formula (8), X is an alkanesulfol group or a benzenesulfol group.

[6]

The production method according to any one of [1] to [5], wherein the nucleic acid derivative and the z- or phenyldiaziridine-added nucleic acid derivative have a label.

[7]

The production method according to [5], wherein the label is piotin, a radioisotope, and Z or a fluorescent substance. A phenyldiaziridine-added nucleic acid derivative represented by the general formula (D).

[Chemical 8]

In general formula (D), R is

Four

[Chemical 9]

Tens — S tens CH ₂ -n, V 'n or

N is an integer of 1 to 6, X and y are independently 0 to: an integer of L00, and N is a group represented by the following general formula (B).

1 2 group, R is OH group or adjacent nucleotide, B is adenine, guanine,

Three

[9]

The derivative according to [8], wherein the phenyldiaziridine-added nucleic acid derivative has a label.

[Ten]

The derivative according to [9], wherein the label is piotin, a radioisotope, and Z or a fluorescent substance [11]

Reaction of a nucleotide derivative represented by the following general formula (E) or (F) with a ferrodiaziridine compound having a reactive group with a thiol group in the compound represented by the general formula (E) or (F) A process for producing a phenyldiaziridine-added nucleotide derivative.

[Chemical 11]

()

In general formula (E), R is adenine, guanine, cytosine, uracil, thymine, and their derived physical strength group power, any base selected, n is 0, 1 or 2, and general formula (F) R and R are independently nicotinamide adenine nucleotide and its phosphate,

1 2

And flavin mononucleotide, sugar phosphate, sugar nucleotide, coenzyme A, and phospholipid.

[12]

The production method according to [11], wherein the phenyldiaziridine complex is a compound represented by the general formula (C).

[Chemical 12]

In general formula (C), R is

Five

[Chemical 13]

R ₆ ten CII ₂ R ₇ - S tens of CII ₂ door

^N ,, n

And

R is a halogen atom or a sulfonate residue,

6

7

Yes,

R is H or an alkyl group having 1 to 6 carbon atoms,

8

n is an integer of 1-6.

[13]

The method according to [12], wherein the phenyldiaziridine complex is a compound represented by the following formulas (1) to (10).

[Chemical 14]

(Hal represents halogen.)

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), an unsubstituted or substituted alkyl group having 1 to 6 carbon atoms, and R 'is H, an unsubstituted or substituted alkyl carbo yl, alkyl carbo-loxy having 2 to 6 carbon atoms, or Is an arylcarboxyl group, and in formula (8), X is an alkanesulfol group or a benzenesulfol group.

[14]

A ferradiaziridine-added nucleic acid derivative produced by the method according to any one of [1] to [7], a ferradiaziridine-added nucleic acid derivative according to any one of [8] to [10], or [11] to [ 13] or the nucleotide derivative produced by the method according to item 1 and the analyte protein are mixed under conditions that allow interaction,

The resulting mixture is irradiated with light to produce a ferradiaziridine-added nucleic acid derivative or nuclease. The diaziridine group contained in the oxide derivative is reacted with the protein to form a conjugate of the above-mentioned ferradiaziridine-added nucleic acid derivative or nucleotide derivative and the protein,

Subjecting the resulting conjugate to electrophoresis,

Protein analysis method by electrical mobility shift assembly.

[15]

The method according to [14], wherein the electrophoresis is performed under denaturing conditions.

[16]

A ferradiaziridine-added nucleic acid derivative produced by the method according to any one of [1] to [7], a ferradiaziridine-added nucleic acid derivative according to any one of [8] to [10], or [11] to [ 13], the nucleotide derivative produced by the method described in any one of the above and the analyte, the protein, are mixed under conditions that allow interaction.

The resulting mixture is irradiated with light to react the diaziridine group contained in the phenyldiaziridine-added nucleic acid derivative or nucleotide derivative with the protein, thereby binding the fertilaziridine-added nucleic acid derivative or nucleotide derivative to the protein. Form the body,

The resulting conjugate is subjected to electrophoresis,

Separating the conjugate, then

The separated conjugate is treated with an alkaline solution to dissociate the conjugate,

Recovering dissociated proteins and Z or nucleic acid derivatives or nucleotide derivatives,

Protein preparation method.

[17]

The method according to [16], 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 occurs very quickly with light near 360 nm. Also, this light is almost against normal biomolecules such as nucleic acids such as DNA and proteins. Not absorbed. 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 multiple binding proteins.

[0010] Further, the photoreactive group in the photoreactive compound and the nucleic acid can be cleaved, and this cleaving reaction is used to bind from the complex of the photoreactive compound (nucleic acid derivative) and the binding protein. Only the protein can be extracted.

BEST MODE FOR CARRYING OUT THE INVENTION

[0011] [Method for producing phenyldiaziridine-added nucleic acid derivative (1)]

The method (1) for producing a phenyldiaziridine-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, and a phenyl group having a reactive group with the thiol group of the thiophosphate group. Reacting with a diaziridine compound.

[0012] 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).

[Chemical 15]

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 is also applicable to a nuclear acid derivative in which the sum of X and y exceeds 200.

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

[Chemical 16]

(B)

In general formula (B), R is an OH group or an adjacent nucleotide. Applicable N is nucleic acid

1

R is an OH group when at the end of the derivative, otherwise R is adjacent

1 1 nucleotide (N). R is hydrogen or an OH group. When R is hydrogen, the nucleus

twenty two

The acid derivative is DNA, and when R is an OH group, the nucleic acid derivative is RNA. 1 bottle

2

Nucleic acid derivatives: Nucleotide in which R is hydrogen (N) and R is OH

twenty two

Chid (N) can be mixed. R is an OH group or adjacent nucleotide. The

Three

R is an OH group when the corresponding N is the end of the nucleic acid derivative, otherwise

Three

R is the adjacent nucleotide (N). B is adenine, guanine, cytosine, ura

Three

Sil, thymine bases and base derivatives. The derivative of a base is, for example, a force that can be 5-methylcytosine, N6-methyladenine, 5-hydroxymethylcytosine, inosine and the like, but is not limited thereto. B is arbitrarily selected from the above bases independently for each nucleotide (N), and can constitute nucleic acid derivatives having various base sequences.

[0016] X is S (sulfur) or O (oxygen). X in normal nucleotide (N) is O (oxygen) and constitutes a phosphate group. A phosphate group in which X is s (sulfur) is a thiophosphate group. In the general formula (A), the nucleotide (N) represented by N—SH represents SH of the thiophosphate group in the general formula (A). N—Nucleotides other than SH (N) may include nucleotides (N) in which X is S (sulfur). A nucleotide (N) in which X is S (sulfur) can be introduced with a phenyldiaziridine group according to the method of the present invention. It is possible to introduce a ferrodiaziridine group into.

[0017] The nucleic acid derivative may have a label. The label can be a known label used for nucleic acid derivatives. Such labels can include, for example, piotin, radioisotopes, and Z or fluorescent materials. However, it is not limited to these substances. The introduction of the label into the nucleic acid derivative can be performed by a known method described in the following document.

b. Agrawal; C. and hnstodoulou; MJ Gait (1986) Efficient methods for attaching non— ra dioactive labels to the 5 'ends of synthetic oligodeoxyribonucleotides Nucleic Acids Res. 14: 6227-6245. “Nonisotopic DNA probe techniques” (ed. LJ Kricka) Acade mic Press, New York

[0018] 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, T.

G. (1991) Phosphorothioate oligonucleotides: chemistry, purification, analysis, scale —up and future directions.Anticancer Drug Des. 6: 539—568. And Caruthers, M.

H.; Beaton, G .; Wu, J.V .; Wiesler, W. (1992) Chemical synthesis of deoxyoligonucle otides and deoxyoligonucleotide analogs. Methods Enzymol. 211: 3—20.

[0019] The ferrulediaziridine compound can be, for example, a compound represented by the general formula (C).

[Chemical 17]

[0020] In the general formula (C), R is

Five

[Chemical 18]

Ma

[0021] In the above formula, R represents a halogen atom (for example, Cl, Br, 1) or a sulfonate ester residue.

6

It is a group. Specifically, the sulfonic acid ester residue can be a methanesulfol group or a toluenesulfol group. R is an alkylthiosulfol having 1 to 6 carbon atoms Group or an alkylthio group having 1 to 6 carbon atoms. The alkylthiosulfol group is RSO

2

R can be, for example, methyl, ethyl, propyl or butyl. The alkylthio group is represented by RS— and R can be, for example, methyl, ethyl, propyl or butyl. R can be H or an alkyl group having 1 to 6 carbon atoms;

8

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.

[0022] The phenyldiaziridine compound represented by the general formula (C) can be, for example, a compound represented by the following formula (1).

[Chemical 19]

[0023] 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 a phenyldiaziridine compound of the formula (2) or the formula (7).

[0024] The phenyldiaziridine compound represented by the general formula (C) can be, for example, a compound represented by the following formula (2).

[Chemical 20]

The compound represented by the formula (2) has halogen (Hal) and diazirine as reactive functional groups. Halogen (Hal) is a thiol group of a thiophosphate group of a nucleic acid derivative having a thiophosphate group. react. 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. Bioche m., 1985, 144, 132-141].

[0026] In the existing synthesis method, a functional group which is a key at the time of introduction into a ligand is previously incorporated on the file skeleton, and a diazirine skeleton that is a photoreactive group is constructed. For example, the halogenated derivatives (2a) and (2b) of ferrodiazirine shown in Scheme 1 below are extremely useful intermediates for the synthesis of various photoreactive probes. In the synthesis methods that have been reported so far, all the functional groups required for the halogeni are synthesized in a multi-step manner because they are assembled together prior to the construction of the diazirine skeleton.

[0027] [Chemical 21]

Scheme 1 (Synthesis of phenyldiazirine compounds with necessary functional groups)

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

[Chemical 22]

[0029] The compound represented by the formula (3) is described in Non-Patent Document 6, M. Kaneda, Y. Sadakane, Y. Hatanaka,

(2003) A Novel Approach for Affinity-Based screening of Target Specinc Ligands: Application of Photoreactive D-Glyceraldehyde-3 -phosphate Dehydrogenase. Bioconjugate Chem. 14, 849-852.

[0030] The phenyldiaziridine compound represented by the general formula (C) can be, for example, compounds represented by the following formulas (4) to (10).

[0031] [Chemical 23]

(4) (5) (6) (7)

In formulas (4), (5), (6), and (9), R is hydrogen), an unsubstituted or substituted alkyl group having 1 to 6 carbon atoms, and in formula (10), R is hydrogen ), Unsubstituted or substituted alkyl having 1 to 6 carbon atoms R ′ is H; an unsubstituted or substituted alkyl carbo group having 2 to 6 carbon atoms such as acetyl group; tert-butoxycarbol (Boc) group, 9-fluorenylmethoxycarbo- An unsubstituted or substituted alkylcarboxoxy group having 2 to 6 carbon atoms such as an Fmoc group; or an arylcarboxoxy group such as a benzoyl group, in which X is methanesulfol, etc. A benzenesulfur group such as p-toluenesulfol. 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.

[0033] As shown in the following scheme 2, the phenyldiazirine derivatives represented by the formulas (4) to (10) start from unsubstituted phenyldiazirine (11), which can be synthesized most simply and in large quantities. Thus, an intermediate (12) in which (11) is directly modified with an aldehyde group is synthesized, and the aldehyde group of this intermediate (12) is further modified to produce a phenotype represented by formulas (4) to (10). Enildiazirine derivatives can be synthesized.

[0034] [Chemical 24]

[Direct modification of phenyldiazirine]

Ferradiazirine is known as an excellent photoreactive group because it is stable to various synthetic 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. Synthetic complexity at that stage was a problem. First, we developed a method for easily synthesizing a useful intermediate aldehyde (12) using (11), which can be synthesized in large quantities, and pioneered a new route through which various useful diazirines can be easily derived. So far, (12) has been reported as a low-yield synthesis method through 7 steps [JM Delfino, SL Schreiber, and FM Richards, J. Am. Chem. Soc, 1993, 115, 3458-3474] ₀ However, in the present invention, instead of this synthesis method, a completely new approach to obtain aldehyde (12) in one step by directly modifying phenyldiazirine (11) is used (Scheme 3).

[0036] [Chemical 25]

Scheme 3 Direct modification of phenyldiazirine

[0037] Until now, CI CHOCH was reacted with ferrodiazirine in the presence of GaCl in TFA-CH C1.

2 3 3 2 2 was able to obtain the aldehyde form (12) at a yield of only 5% [U. Kempin, Y. Kanaoka, and Y. Hatanaka, Heterocycles, 1988, 49 , 465-468.]. During this experiment, it was surprising that the raw material, CI CHOCH, decomposed immediately after the reaction started.

twenty three

As a result of investigating the cause, when CI CHOCH is added to TFA, decomposition occurs.

twenty three

As a result of examining organic acids, CI CHOCH was found to be in trifluoromethanesulfonic acid (TfOH).

twenty three

And found to be stable. Under these conditions, Lewis acids are A1C1, ZnCl, SbCl, SbCl,

3 2 3 5

SnCl, SnCl, Ζη (ΟΤί), AgOTf, Gd (OTi), Pr (OTi), Sm (OTi), Υ (ΟΤί), Yb (OTi),

2 4 2 3 3 3 3 3 When various Lewis acids were examined, formation of the desired aldehyde (12) was confirmed on TLC. Table 1 shows the yields for GaCl, FeCl, SbF, and TiCl.

3 3 5 4

[0038] [Table 1] Table 1 Examination of Lew is acid

Lew i s acid Y i e l d (%)

GaC I ₃ 31

FeC I ₃ 47

SbF ₅ 80

T i CI ₄ 80

[0039] According to this method, a functional group useful for synthesis can be easily introduced onto the aromatic ring of phenyl diazirine, which has heretofore been difficult to modify directly. It is considered to be particularly important in terms of being easily convertible to various functional groups.

[0040] [Application to simple asymmetric synthesis of ferrolanine derivatives having a diazirine group]

The aldehyde compound (12), which can be easily obtained by the Friedd-Crafts reaction, is further reduced by reducing the aldehyde group to the benzyl alcohol compound (13), and the hydroxy group is converted to a rogened compound (14). Can be easily converted to useful synthetic intermediates. This halogen compound is used for the synthesis of a phenol (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. So, 106, 7540-7545 (1984); LB Shih, and H. Bayley, Anal. Biochem., 1985, 144, 1 32—141; WG Fishwick, JM Sanderson, and JBC Findlay, Tetrahedron Let t., 1994, 35, 4611-4614.] This is a very multi-step synthesis method involving diazirine synthesis. Here, the aldehyde (12) force is also started, and (13) is obtained by reduction, and then (14) is obtained by halogenation. (14) Kasaka et al. (15) can be easily asymmetrically synthesized by several steps of reaction (Scheme 4). In Scheme 4 below, (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 ( In 10), R and R ′ are hydrogen atoms (H).

[0041] [Chemical 26]

[0042] Reaction of a nucleic acid derivative with a thiol-reactive phenydiaziridine compound

In the method (1) of the present invention, a ferrodiaziridine compound having a thiol-reactive group and a nucleic acid derivative are mixed at a molar ratio of, for example, about 50: 1 to 100: 1. Further, this mixture is mixed with diisopropylethylamine in a molar ratio of 50 to 100 times with respect to the nucleic acid derivative, and the reaction can be carried out in dimethyl sulfoxide at 37 ° C. However, N-methylmorpholine, triethylamine and the like can be used in place of diisopropylethylamine. As a reaction solvent, methanol, dimethylformamide or the like can be used instead of dimethyl sulfoxide. The reaction temperature can include 37 ° C, for example, in the range of 4 to 70 ° C.

[0043] The concentration of the ferrodiaziridine compound is suitably in the range of 0.1 to 0.5 mM, for example. The reaction time can be a force depending on the type of ferrodiaziridine compound, for example, several tens of minutes, several hours. It is preferable to store in liquid nitrogen after the reaction so that no excessive reaction occurs! The product, a ferrodiaziridine-added nucleic acid derivative, can be purified by, for example, high performance liquid chromatography. In addition to high performance liquid chromatography, it can be purified by capillary electrophoresis, for example.

[0044] Examples of the above reaction are shown in the following schemes.

Scheme 5 is an example in which the compound (2a) is used as a ferrodiaziridine 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. The thiol hydrogen of the thiophosphate group and Br of the compound (2a) form HBr, and by de-HBr, the compound (2a) and the nucleic acid derivative (Al) are combined to form a phenyldiaziridine-added nucleic acid derivative (D1). Generate.

[0045] [Chemical 27]

[0046] Scheme 6 is an example in which the compound (3) is used as a ferrodiaziridine compound. The nucleic acid derivative (A2) is an example having a thiophosphate group at the terminal nucleotide of the nucleic acid derivative. The thiol atom of the thiophosphate group nucleophilically reacts with the thiosulfonate group (MeS O S—) of the compound (3).

twenty two

A disulfide bond is formed to produce a phenyldiaziridine-added nucleic acid derivative (D2). [0047] [Chemical 28]

Scheme 6

(A 2) (D

[0048] Scheme 7 is an example in which compound (2a) is used as a ferrodiaziridine compound. The nucleic acid derivative (A2) is an example having a thiophosphate group at the terminal nucleotide of the nucleic acid derivative. The hydrogen of the thiol of the thiophosphate group and Br of the compound (2a) form HBr, and by de-HBr, the compound (2a) and the nucleic acid derivative (A2) are combined to form a ferrodiaziridine-added nuclear acid derivative ( D3) is generated.

[0049] [Chemical 29] Scheme 7

(2a) (Λ 2) (D 3)

[0050] Scheme 8 is an example in which the compound (1) is used as a ferrodiaziridine compound. The nucleic acid derivative (A2) is an example having a thiophosphate group at the terminal nucleotide of the nucleic acid derivative. The hydrogen of the thiol of the thiophosphate group is transferred to the compound (1), and the compound (1) and the nucleic acid derivative (A2) are combined to produce a phenyldiaziridine-added nucleic acid derivative (D4).

[0051] [Chemical 30]

Scheme 8

[0052] The ferrodiaziridine-added nucleic acid derivative can be a compound represented by the general formula (D). The present invention includes a fermenteraziridine-added nucleic acid derivative itself as well as a method for producing a ferraziaziridine-added nucleic acid derivative.

[Chemical 31]

N _x NN _y

In general formula (D), x, y, and N are the same as defined in general formula (A). R can be any one of the following general formulae. N in the following general formula

Four

Can be, for example, an integer from 1 to 6.

[0054] [Chemical 32]

[0055] The fermentiaziridine-added 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, piotin, radioisotopes, and Z or fluorescent materials. However, it is not limited to these substances.

[0056] [Method for producing phenyl diaziridine-added nucleotide derivative (2)]

The method (2) for producing a phenyldiaziridine-added nucleotide derivative of the present invention comprises a nucleotide derivative represented by the following general formula (E) or (F) and a compound represented by the general formula (E) or (F): And reacting a ferrodiaziridine compound having a reactive group with a reactive thiol group. [Chemical 33]

[0057] In general formula (E), R is any base selected from the group consisting of adenine, guanine, cytosine, uracil, thymine, and derivatives thereof, and a derivative of the base includes, for example, 5-methylcytosine N6-methyladenine, 5-hydroxymethylcytosine, inosine, and the like. n is 0, 1 or 2.

In general formula (F), R and R are independently nicotinamide adenine nucleotide and its

1 2

Phosphorus oxide and flavin mononucleotide, sugar phosphate, sugar nucleotide, coenzyme A, and phospholipid strength are selected.

[0059] Examples of the phosphoric acid oxide of nicotinamide adenine nucleotide include nicotinamide adenine dinucleotide phosphate. Examples of flavin nucleotides include flavin adene 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, Some of them are branched sugars, aminouronic acids, ketoses, sulfate sugars and oligosaccharides. Examples of coenzyme A include acetyl acetylenzyme A. Examples of the phospholipid include glyceguchi phospholipid, phosphatidylcholine (lecithin), phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol (cardiolipin), and sphingophospholipid.

[0060] The ferrodiaziridine 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.

[Chemical 34]

[0061] [Protein Analysis Method Using Electrical Mobility Shift Access]

The present invention includes a method for analyzing a protein by electrical mobility shift assay. In this analysis method, the phenyldiaziridine-added nucleic acid derivative produced by the method (1) of the present invention and the phenyldiaziridine-added nucleic acid derivative of the present invention, or the nucleotide derivative produced by the method (2) are used.

[0062] The analysis method includes the following steps.

(a) A mixture of a fermentiaziridine-added nucleic acid derivative or nucleotide derivative and a protein as an analyte under conditions that allow interaction.

(b) The obtained mixture is irradiated with light to react the diaziridine group contained in the phenyldiaziridine-added nucleic acid derivative or nucleotide derivative with the protein, and the phenyldiaziridine-added nucleic acid derivative or nucleotide derivative and protein To form a conjugate.

(c) The obtained conjugate is subjected to electrophoresis.

[0063] 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 (eg, DNA) is used for interaction with proteins after being purified as necessary. A nucleic acid derivative having a tag such as biotin introduced at the 5 ′ end can be used.

[0064] 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.

[0065] Step (b)

The resulting mixture is irradiated with light to react the diaziridine group contained in the phenyldiaziridine-added nucleic acid derivative or nucleotide derivative with the protein. The light to be irradiated can be, for example, light around 360 nm. Conditions such as the irradiation time vary depending on the intensity of the light source, but can be, for example, several seconds to 30 minutes on ice. By the above light irradiation, a covalent bond is formed between the ferrodiaziridine-added nucleic acid derivative and the protein, thereby linking the two.

[0066] Upon irradiation with light, the diaziridine group of the phenyldiaziridine-added nucleic acid derivative or nucleotide derivative reacts with an arbitrary part of the protein to form a conjugate.

[0067] Step (c)

In step (c), the resulting conjugate is subjected to electrophoresis. Electrophoresis can be normal gel electrophoresis. Specifically, by placing a sample on top of the slab gel and flowing an appropriate current flow rate, the sample flows 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, allowing separation analysis.

[0068] Furthermore, electrophoresis can be performed under denaturing conditions, and it is also 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 a surfactant is also allowed to coexist to make the charge of the sample uniform.

[0069] 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.

[0070] [Protein Preparation Method]

The present invention encompasses a method for preparing a protein. In this preparation method, the method of the present invention ( The ferrodiaziridine-added nucleic acid derivative produced in 1), the ferrodiaziridine-added nucleic acid derivative of the present invention, or the nucleotide derivative produced by the method (2) of the present invention is used.

[0071] The method for preparing a protein includes the following steps.

(d) Mixing with phenyldiaziridine-added nucleic acid derivative or nucleotide derivative under conditions that allow interaction with the protein of interest.

(e) irradiating the resulting mixture with light to react the diaziridine group contained in the phenyldiaziridine-added nucleic acid derivative or nucleotide derivative with the protein, and to add the above-mentioned diazaziridine-added nucleic acid derivative or nucleotide derivative to the protein. To form a conjugate of

(f) separating the conjugate.

(g) The separated conjugate is treated with an alkaline solution to dissociate the conjugate, thereby recovering the protein.

[0072] Steps (d) and (e) can be carried out in the same manner as steps (a) and (b).

[0073] Step (f)

For selection of the conjugate to be separated, for example, a conjugate of a nucleic acid derivative having a 5'-end introduced with a tag such as piotin and a protein 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 piotin is introduced at the 5 ′ end, the conjugate is separated with beads fixed with avidin as a complex with the protein. Capture and implement.

[0074] 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 thio atoms is due to the nucleophile. Transfer the separated complex into a solution adjusted to pH 10.5 with 50 mM phosphoric acid and sodium hydroxide, and leave it at 37 ° C. 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. Scheme 9 below shows the chemical state of the dissociation of the conjugate.

[0075] [Chemical 35] Scheme 9

(A l) (2)

Example

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

[0077] Reference Example 1

Method for synthesizing compound of formula (1)

[Chemical 36]

[0078] N- [4- [3-trifluoromethyl) -3H-diazirin-3-ylJbenzyl] maleimide (1)

4- [3- (Tnfluoromethyl)-ύΗ- diazirm- 3-yl] benzyl alcohol (13) (750 mg, όΛ5 mmol) is dissolved in 28 mL of THF, and triphenylphosphine (1.05 g, 3.9 mmol), maleimide (389 mg, 3.97 mmol) was added, and the mixture was 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%) NMR-NMR (CDC1)

Three

: 7.37 (d, 2H, J = 7.3 Hz) 7.19 (d, 2H, J = 7.3 Hz) 6.72 (s, 2H) 4.67 (s, 2H).

[0079] Reference Example 2-1

[Chemical 37]

[0080] 4- [3- (Trifluoromethyl) -3H-diazirin-3-y (12)

3— (Phenyl) — 3— (trifluoromethyl) — 3H— diazirine (11) (1.86 g, 0.01 mol) is zero. C in CI CH

2

Dissolved in OCH (3.45 g, 0.03 mol). TfOH (1.

Three

77 mL, 0.02 mol) and TiCl (2.85 g, 0.015 mol)

Four

It was. When the salty hydrogen generation stopped, it became an orange liquid. After 1 hour at room temperature, 500 mL of hexane was added to the reaction mixture, and crushed ice was slowly added under zero ice to decompose excess reagent, followed by neutralization by slowly adding solid sodium carbonate. did. 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 C1 = 2: 1) to obtain a yellow oily substance (12).

twenty two

[0081] Yield 1.71 g (80%)

1H-NMR (CDC1)

Three

σ: 10.04 (s, 1H), 7.91 (d, 2H, J = 8.3 Hz) 7.35 (d, 2H, J = 8.3 Hz).

[0082] Reference Example 2-2

[Chemical 38]

[0083] 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 a compound in which R and R ′ are hydrogen atoms (H) in the above (10).

[0084] 4- [3- (Trifluoromethyl) -3H-diazirin-3-yl] benzyl alcohol (13)

Compound (12) (3.3 g, 15.4 mmol) was dissolved in 7 mL of EtOH and stirred at 0 ° C with NaBH (0.

Four

626 g, 16.5 mmol) suspended in EtOH 7 mL was collected. After the addition was completed, the mixture was returned to room temperature and stirred for 4 hours. To this, 1M HC1 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).

[0085] Yield 3.21 g (96%)

JH-NMR (CDC1)

Three

σ: 7.39 (d, 2Η, J = 8.5 Hz), 7.19 (d, 2H, J = 8.5 Hz), 4.71 (s, 2H), 1.87 (bs, 1H) [0086] Reference Example 2-3

3- [4- (Bromomethyl) phenyl]-3- (trifluoromethyl)-3H- diazirme (14)

Compound (13) (344.0 mg, 1.59 mmol) was dissolved in 2 mL of CH CI and CBr (658.2 mg, 1.985

2 2 4

mmol). It was cooled to 0 ° C and Ph P (474 mg, 1.807 mmol) was slowly added. Until room temperature

Three

After stirring for 1 hour, the mixture was purified by silica gel column chromatography (Hexane) to obtain a colorless oily substance (14).

[0087] Yield 401.8mg (90%)

JH-NMR (CDC1)

Three

σ: 7.42 (d, 2Η, J = 8.5 Hz), 7.17 (d, 2H, J = 8.5 Hz), 4.46 (s, 2H).

[0088] Reference Example 2-4 L- 4 [3- (Trifluor omethyl)-3 H- diazirin- 3-yl] phenylalanine (15)

tert-Butylglycinate benzophenone imine (1.136 g, 3.839 mmol) and asymmetric catalyst 0 (9)

[9]

— Ally N—9— Anthracenylmethylcinchonidium bromide (0.2127 g, 0.351 mmol, 0.1 eq) was dissolved in 9 mU of CH CI. Add compound (14) (1.419 g, 5.085 mmol, 1.5 eq.)

twenty two

The mixture was stirred at room temperature under a nitrogen gas atmosphere. While cooling the reaction mixture to -78 ° C, add 2-tert-Butyl imino— 2-methylamino— 1,3— dimethylperhirao— 1,3,2— diazaphosphorine (1.4 g, 5.139 mmol, Aldrich) in a few seconds. The solution was added dropwise and reacted 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.

[0089] Yield 1.89 g (95%)

JH-NMR (CDC1)

Three

σ: 7.57 (d, 2Η, 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).

[0090] 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 C1 and extracted three times with water. The aqueous layer was lyophilized to a white solid (

twenty two

15) was obtained.

[0091] Yield (as TFA salt) 53.4 mg (91%)

1H-NMR (CD OD)

Three

σ: 7.46 (d, 2Η, 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, lH), 3.10 (d , 1H, J = 4.5, 8.2 Hz).

Optical purity (e.e.)

(5) was derived into Fmoc form, and its optical purity was confirmed by HPLC. Dissolve the appropriate amount in MeOH, then elute with a chiral column (Sumichiral OA-3300 2.5 μm 4.6 mm x 25 cm) at 0.01 M Ammonium Acetate / MeOH at a flow rate of 1 mL / min. Detected by absorption. ee = 98%

[a] + 22.4 ° (c = 0.52, EtOH)

D

[0092] Example 1

As a nucleic acid derivative, a 4-mer DNA guanine and cytosine phosphate having a S-row of adenine, guanine, cytosine, and thymine represented by compound (A3) in the following scheme 10 is a thioester. The thing (AGsCT) was used. This compound was synthesized by a known method described in the following literature. Zon, G .; Geiser, TG (1991) Phosphorothioate oligon ucleotides: chemistry, purification, analysis, scale-up and iuturedirections .Antiance r Drug Des. 6: 539-568. JV; Wiesler, W. (1992) Chemical synthesis of deoxyoligonucleotides and deoxyoligonucleotide analogs.Methods Enzymol. 211: 3-20.

[0093] [Chemical 39]

(A 3) (2 a) (D 5)

[0094] The DNA containing 0.05 mM AGsCT (A3) thio atom was mixed with compound (2a) in scheme 3 in a molar ratio of 100 times and diisopropylethylamine in a molar ratio of 200 times. . The reaction was carried out at 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.

[0095] The chart shown in FIG. 1 is obtained by high performance liquid chromatography when the reaction is performed at 37 ° C. This is an analysis example. Chart 1 is before the reaction, and charts 2, 3, 4, and 5 are after 1, 2, 4, and 24 hours after the reaction, respectively. The numbers at the top of the peak indicate the retention time. The peak around 6.5 minutes is the compound (A3) in Scheme 10 of the raw material, and the peak around 7.5 minutes is the compound (D5) in the product scheme 10. .

[0096] As a result of fractionating the peak portions corresponding to this compound (A3) and compound (D5) and performing mass spectrometry analysis, 1191 (M + H +) was obtained for compound (A3), and 1388 (M) was obtained for compound (D5). + H +) measurement result was obtained. These are in good agreement with the theoretical mass values 1191.22 (M + H +) and 1388.26 (M + H +) of compound (A3) and compound (D5), indicating that compound (D5) can be produced from compound (A3).

[0097] Graphs (1), (2), and (3) shown in Fig. 2 show the reaction time and product compound (D5) when the reaction was carried out at 20 ° C, 37 ° C, and 56 ° C, respectively. ) Yield. Yield is good at the reaction temperature of 37 ° C in graph (2). The maximum yield is obtained by the reaction for about 2 hours.

[0098] 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.

[0099] The reaction temperature was fixed at 37 ° C, and the reaction yield when the concentration of the compound (1) in Scheme 3 as a raw material was 0.023, 0.045, 0.182, 0.455 mM was shown in graphs (1), ( Shown in 2), (3) and (4). The reaction is faster at high concentrations of raw material, but there is no change at around 0.2 mM. Although the preferred 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 O.lmM.

[0100] Example 2

[Chemical 40]

Scheme 1 1

(1) (Λ 2) (D 4) [0101] 21mer RNA (A2) thiophosphorylated at the 5 'end and a ferrodiaziridine compound represented by formula (1) were prepared in dityl sulfoxide to final concentrations of 0.025 mM and 20 mM, respectively. did. Further, diisopropylethylamine was mixed so that the final concentration was 20 mM and 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. 4.

[0102] Chart (1) shown in Fig. 4 shows the analysis result of the raw material (A2) by high performance liquid chromatography. The peak force corresponding to (a) in the chart is a 2 lmer RNA (A2) in which the 5 'end of the raw material is thiophosphated. Chart (2) above is a similar analysis of product (D4). The peak (b) in the chart is a newly appearing peak and was confirmed to be (D4) as a result of mass spectrometry.

[0103] Example 2

[0104] [Chemical 41]

Scheme 1 2

(A 1) (D

[0105] 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 of the compound represented by the formula (2a) and the 5 'end are piotinated and one of the nucleic acids in the DNA sequence is thiophosphate, Dissolved in dimethyl sulfoxide to final concentrations 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-speed 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) Phos phorotnioate oligonucleotides: chemistry, purincation, analysis, scale-up and iuturedi rections .Anticancer Drug Des. 6: 539-568. And Caruthers, MH; Beaton, G .; Wu, JV; Wiesler, W. (1992) Chemical synthesis of deoxyoligonucleotides and deoxyoligo nucleotide analogs.Methods Enzymol.211: 3— 20.

[0106] 0.1 μg of the product represented by the chemical formula (Dl), HeLa cell nuclear extract g, 25 mM HEPE S-Na ゝ 50 mM NaCl, 5 mM DTT ゝ ImM EDTA, 0.05% Triton X-100, 10% glycerol, _{2 μ § poly (dI 'dC} ), was dissolved in a mixed solution of 20 L of lOO ^ g / mL BSA, for 1 hour to stand in ice. When binding was inhibited, 10 to 100-fold molar amount of the competing DNA to be inhibited was allowed to coexist.

[0107] The tube containing the above 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 irradiation, 5 L of a denaturing solution for electrophoresis was added, and the mixture was allowed to stand at room temperature for 30 minutes or more. Figure 5 shows a schematic diagram of this operation.

[0108] 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, and the separation gel was immersed in a fixing solution and transferred from the gel to the membrane using a semi-dry blotting apparatus. The membrane was blocked with 1.0% (w / v) force zein / PBS for 1 hour at room temperature, and then added with 0.1% (v / v) Strept-avidin HRP / PBS-T and shaken at room temperature for 30 minutes. After washing the membrane several times, peotine was detected with a chemiluminescent reagent.

[0109] Piotin is originally bound to photoreactive DNA. When photoreactive DNA alone is analyzed by electrophoresis using a 10% polyacrylamide gel, the molecular weight is as low as about 5000, so it flows out of the gel. Piotin 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.

[0110] The lanes in the analysis example shown in Fig. 6 will be described. Lane “Μ” 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 only the photoreactive DNA was used without the nuclear extract. Lane “2” is an experiment in which the nuclear extract and photoreactive DNA were added and irradiated 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 a 25-fold molar amount of DNA obtained by mutating part of the DNA sequence.

[0111] Comparing lanes “1” and “2” demonstrates the ability to analyze many binding proteins simultaneously. Inhibition experiments in lanes “3” and “4” indicate that these complex formations are specific to the DNA sequence.

[0112] Example 4

[Example of cutting method]

An example is shown in which the same reaction as in the scheme shown in Example 3 was performed using a sample prepared by introducing diazirin into a 4-mer DNA fragment of AGsCT. The photoreactive DNA was dissolved in a solution prepared by adding 50 mM phosphate buffer with sodium hydroxide to 10.5 with a pH of 10.5 so that the photoreactive DNA was 10 μ μ. 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.

[0113] Chart (1) above is a high-performance liquid chromatographic analysis using photoreactive AGsCT before reaction as a sample, and the peak (a) in the chart corresponds to it. Chart (2) is an analysis of the sample after reacting for 2 hours by the above method. The peak corresponding to (a) disappears, and the new peak (b) appears. Chart (3) is an analysis of AGsCT without diazirine as a sample, and the peak in (c) corresponds to it. Since the retention times of the peak in (b) and the peak in (c) are almost the same, the peak in (b) is expected to be AGsCT with diazirine cleaved. As a result of fractionating this (b) peak and analyzing it by mass spectrometry, it was shown that it was A GsCT without diazirine.

[0114] Example 5

[Example of cutting]

In the same manner as in the scheme shown in Example 3, Biotin-TGT ATGsCAAATAAGG (SEQ ID NO: 1) having a 5'-end of piotin was used to produce photoreactive DNA. Fig. 8 shows a schematic diagram of the example below.

[0115] The photoreactive DNA is placed in a polypropylene tube (Fig. 8 (1)) and fixed to the tube surface by light irradiation (Fig. 8 (2)). After fixation, the DNA is removed by washing (Fig. 8 (3)), and the presence or absence of piotin is confirmed by fluorescent light emission with streptavidin-HRP enzyme (Fig. 8 (4)). The cleavage process is performed after the operation shown in Fig. 8 (3). I confirmed that it was removed from the surface of the groove.

[0116] The detailed experimental procedure is as follows.

1. Add 50 μL of fluorescently reactive DNA (in water) to a PCR tube and dry.

2. Fix the DNA to the tube by photoreaction (360 nm, 5 min).

Wash with 100% of 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. Cutting P-S under cutting conditions

Condition 1 Add 100 μL of the solution adjusted to pH 10.5 with> 50 mM phosphate buffer + NaOH and leave at 37 ° C for 2 hours.

<Condition 2> Add 50 μM 50 mM Acetate—Na buffer (pH 3.0) 100 mM NaCl, and leave at 37 ° C overnight (about 15 hours).

Leave 6.1% casein PBS 100 / z L for 1 hour at 37 ° C. (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), 100 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.

[0117] One of the chemiluminescence detection examples in Fig. 9 is one in which cleavage treatment is not performed, and biotin DNA is immobilized on the tube surface. Detection Example 2 does not perform photoreaction and does not perform the fixed 匕 process. Detection Example 3 is an example of cleavage treatment, in which a 50 mM phosphate buffer solution was placed in a solution adjusted to pH 10.5 with sodium hydroxide. The tube surface force by the cutting process also indicates that the Piotin DNA was removed.

[0118] Example 6

[Example of separation of binding protein]

An example is shown in which a protein that binds to the DNA sequence is separated using photoreactive DNA. In the same scheme as in Example 3, Biotin-TGTATGsC AAATAAGG (SEQ ID NO: 1) having a 5'-end that was piotinated was synthesized with a photoreactive DNA, and the target product was purified by high performance liquid chromatography. Photoreactive DNA is 10 / z M and binds to this DNA sequence. Protein 7.8 μg and 25 mM HEPES—Na, 50 mM NaCl, 5 mM DTT ゝ ImM EDTA, 0.05% Triton X-100, 10% glycerol, 2 g poly (dI'dC), 100 μg / mL BSA It was dissolved in 20 μL of the solution and allowed to stand in ice for 1 hour.

[0119] A tube containing the above sample was floated on ice water, and irradiated with 360 nm light 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 performed in a solution prepared by adjusting 50 mM phosphate buffer to 10.5 with sodium hydroxide. The supernatant was collected, separated by electrophoresis, and the protein was stained with silver stain. The result is shown in FIG.

[0120] 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 had been cleaved as described above as a sample, lane “2” used the sample that had been cleaved without POU protein, and lane “3” POU protein was used as a sample. 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”. A cleavage occurred between the POU protein and DNA, indicating that the protein captured by the photoreaction was recovered.

[0121] Example 7

[Introduction of diazirine into GTP-y-S, ADP-β-S]

[0122] Diazirine was introduced into the phosphorothioates of GTP-γ-S and ADP-β-S shown below.

Scheme 1 3

[0123] <Operation>

• Creation of photoreactive GTP-y-S

Prepare a sample to a final concentration of 1 mM GTP-y-S, 100 mM phosphate buffer (pH 7.0), 50 mM 4- [3H- [3-trifluoromethyl] diazine-3-yl] benzyl maleimide, Leave at 37 ° C for 20 hours. After the reaction, it was analyzed and isolated by HPLC, and the molecular weight was measured by MASS.

[0124] · Creation of photoreactive ADP-β-S

Prepare a sample to a final concentration of 1 mM ADP-jS-S, 100 mM phosphate buffer (pH 7.0), 50 mM 4- [3H- [3-trifluoromethyl] diazine-3-yl] benzyl maleimide, Placed at 37 ° C for 18 hours. After the reaction, the product was analyzed and isolated by HPLC, and the molecular weight was measured by MASS. The results are shown in FIG.

[0125] From the HPLC results, a new product peak was confirmed at 14.2 min for GTP y -S and 14.0 min for ADP-β -S in the diaziridine introduction reaction. This peak was collected and the molecular weight was measured by MASS.

[0126] [Table 2] MASS results of peaks after diazirine introduction reaction to GTP-rS and -S

[0127] From the results of molecular weight measurement, it was confirmed that diazirine was introduced into GTP-γ-S and ADP-β-S. The yields were 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.

[0128] Example 8

Photoreactive label of photoreactive GTP and Ra-Ras

[Chemical 43]

[0129] 40 ng H—Ras ゝ 10 mM diazirine—GTP ゝ 10 mM MgCl, 50 mM Tris—HCl (pH 7.4)

2

A 1 mL sample 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 an aqueous solution containing 1 mM N-ethylmaleimide (NEM), 1 mM tri-n-butylphosphine, 0.5 M phosphate buffer (pH 7.0), and 10% ethanol to the sample, and 20 ° C under argon. Incubated for 20 hours. 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, the ammonia was removed by bubbling nitrogen, and 40 mL of an aqueous solution containing 1 mM piotinmaleimide 0.5 M phosphate buffer (pH 7.0) and 10% ethanol was added, and 20 under argon. Incubated at C for 20 hours. The sample after the reaction was treated with piotin male on a Sephadex G25 column. The mid was removed and lyophilized. The sample was 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.

[Chemical 44]

Diazirine-GTP

Diazirine-ATP

Example 9

Isolation and detection of optical label Ras using GTP tag

40 ng H-Ras ゝ 10 mM Diazirine-GTP ゝ 10 mM MgCl, 50 mM Tris-HCl (pH 7.4)

2

Prepare 1 mL of sample and incubate 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 of 1 mM piotin-0 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 equal volume (45mL), Fe ³⁺ -IMAC column (50mL min) carrying 〖this ^$. After washing with 0.1 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% aqueous ammonia solution. So 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. Figure 14 shows the reaction scheme.

[0132] † Results 3-5 added GTP for inhibition at this point.

* Result 2 shows 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.

Industrial applicability

[0133] The present invention is useful in a wide range of fields involving nucleic acids and proteins.

Brief Description of Drawings

[0134] [Fig. 1] An example of analysis by high performance liquid chromatography is shown.

FIG. 2 shows the relationship between reaction time and yield.

FIG. 3 shows the relationship between reaction concentration and yield.

[Fig. 4] Shows an example of analysis by high performance liquid chromatography.

[Fig. 5] Schematic diagram of formation of binding protein with covalently linked DNA.

[Figure 6] Shows an example of complex analysis detected by chemiluminescence.

[Fig. 7] An example of analysis by high performance liquid chromatography is shown.

FIG. 8 is a schematic diagram of Example 6.

FIG. 9 shows an example of chemiluminescence detection.

FIG. 10 is an electrophoresis image of the collected supernatant.

[Fig. 11-1] Analysis of introduction reaction of diazirine into GTP-γ-S by HPLC.

[Fig. 11-2] Analysis of introduction reaction of diazirine into ADP-β-S by HPLC.

FIG. 12 shows the results of chemiluminescence detection of the optical label Ras using the optical probe-specific piotine-attached cage in Example 8. 1: Optical label is present 2: Optical label is not present (The light irradiation of the operation * is performed)

[FIG. 13] Isolation and detection results of optically labeled Ras using GTP-tag in Example 9. 1: With optical label 2: Without optical label 3: Equivalent Inhibition with GTP 4: 10 Equivalent Inhibition with GTP 5: 1

00 equivalent GTP inhibition FIG. 14: Reaction scheme in Example 9.

Claims

The scope of the claims

[1] A nucleic acid derivative (provided that at least one of the nucleic acid phosphate groups is a thiophosphate group) is reacted with a thiol group of a thiophosphate group and a phenyldiaziridine hydrate compound having a reactive group. And a method for producing a ferrule-added nucleic acid derivative.

[2] The production method according to claim 1, wherein the nucleic acid derivative is a compound represented by the general formula (A).

[Chemical 1]

IN _V刚,

(A)

SH

[Chemical 2]

1 2

R is an OH group or adjacent nucleotide, B is adenine, guanine,

Three

[3] The production method according to claim 1 or 2, wherein the phenyldiaziridine complex is a compound represented by the general formula (C).

[Chemical 3]

R is a halogen atom or a sulfonate residue,

6

7

Yes,

R is H or an alkyl group having 1 to 6 carbon atoms,

8

n is an integer of 1-6.

4. The production method according to any one of claims 1 to 3, wherein the phenyldiaziridine-added nucleic acid derivative is a compound represented by the general formula (D).

[Chemical 5]

N _x N _y

In general formula (D), x, y, and N are the same as defined in general formula (A), and R [Chemical 6]

And n is an integer of 1-6.

The method according to claim 3, wherein the phenyldiaziridine compound is a compound represented by the following formulas (1) to (10).

[Chemical 7]

6. The production method according to any one of claims 1 to 5, wherein the nucleic acid derivative and the Z- or phenyldiaziridine-added nucleic acid derivative have a label.

[7] The production method according to claim 5, wherein the label is piotin, a radioisotope, and Z or a fluorescent substance.

[8] A phenyldiaziridine-added nucleic acid derivative represented by the general formula (D).

[Chemical 8]

s

N _V NN, y

In general formula (D), R is

Four

[Chemical 9]

Tens CH +-— S tens

, ¹¹ ,

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).

[Chemical 10]

1 2 group, R is OH group or adjacent nucleotide, B is adenine, guanine, Any base selected from the group consisting of tosine, uracil, thymine and derivatives thereof, X is S (sulfur) or o (oxygen).

9. The derivative according to claim 8, wherein the phenyldiaziridine-added nucleic acid derivative has a label.

10. The derivative according to claim 9, wherein the label is piotin, a radioisotope, and Z or a fluorescent substance.

[11] A phenyldiaziridine compound having a nucleotide derivative represented by the following general formula (E) or (F) and a reactive group with a thiol group in the compound represented by the general formula (E) or (F) And a method for producing a phenyldiaziridine-added nucleotide derivative.

[Chemical 11]

1 2

12. The production method according to claim 11, wherein the phenyldiaziridine compound is a compound represented by the general formula (C).

[Chemical 12]

In general formula (C), R is

Five

[Chemical 13]

And

R is a halogen atom or a sulfonate residue,

6

7

Yes,

R is H or an alkyl group having 1 to 6 carbon atoms,

8

n is an integer of 1-6.

13. The method according to claim 12, wherein the phenyldiaziridine compound is a compound represented by the following formulas (1) to (10).

[Chemical 14]

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), an unsubstituted or substituted alkyl group having 1 to 6 carbon atoms, and R ′ is H, an unsubstituted or substituted alkyl carbonyl group having 2 to 6 carbon atoms.

, An alkylcarboxoxy group or an arylcarboxoxy group, and in the formula (8), X represents an alkanesulfol group or a benzenesulfol group.

A phenyldiaziridine-added nucleic acid derivative produced by the method according to any one of claims 1 to 7, claim 8 to: any one of L0, a phenyldiaziridine-added nucleic acid derivative according to claim 1, or A nucleotide derivative produced by the method according to any one of claims 11 to 13 and a protein that is an analyte are mixed under conditions that allow interaction,

The resulting mixture is irradiated with light to react the diaziridine group contained in the fermentiaziridine-added nucleic acid derivative or nucleotide derivative with the protein, and to react the fermenter. Form a conjugate of an aziridine-added nucleic acid derivative or nucleotide derivative and a protein;

Subjecting the resulting conjugate to electrophoresis,

Protein analysis method by electrical mobility shift assembly.

15. The method according to claim 14, wherein the electrophoresis is performed under denaturing conditions.

[16] Phenyldiaziridine-added nucleic acid derivative according to any one of claims 1 to 7, wherein the phenyldiaziridine-added nucleic acid derivative is produced by the method according to any one of claims 1 to 7. Claim 8 to: A derivative, or a nucleotide derivative produced by the method according to any one of claims 11 to 13, and a protein that is an analyte are mixed under conditions that allow interaction,

The resulting conjugate is subjected to electrophoresis,

Separating the conjugate, then

Protein preparation method.

17. The method according to claim 16, wherein the electrophoresis is performed under denaturing conditions.