WO2006120992A1 - Derives d’acide nucleique presentant des fragments de phenyldiaziridine et leur procede de production, derives de nucleotides presentant des fragments de phenyldiaziridine et leur procede de production, procede d’analyse de proteine et procede de preparation de proteine - Google Patents

Derives d’acide nucleique presentant des fragments de phenyldiaziridine et leur procede de production, derives de nucleotides presentant des fragments de phenyldiaziridine et leur procede de production, procede d’analyse de proteine et procede de preparation de proteine Download PDF

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WO2006120992A1
WO2006120992A1 PCT/JP2006/309229 JP2006309229W WO2006120992A1 WO 2006120992 A1 WO2006120992 A1 WO 2006120992A1 JP 2006309229 W JP2006309229 W JP 2006309229W WO 2006120992 A1 WO2006120992 A1 WO 2006120992A1
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nucleic acid
phenyldiaziridine
general formula
acid derivative
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PCT/JP2006/309229
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Japanese (ja)
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Yasumaru Hatanaka
Yutaka Sadakane
Masaki Kaneda
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National University Corporation University Of Toyama
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical

Definitions

  • Phenyldiaziridine-added nucleic acid derivatives and their production methods Phenyldiaziridine-added nucleotide derivatives and their production methods, and protein analysis methods and preparation methods
  • 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.
  • 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.
  • 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.
  • EMSA Electrical mobility shift assay
  • DNA DNA
  • RNA DNA
  • 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.
  • 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.
  • 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. ⁇ 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
  • 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) )
  • 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.
  • 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).
  • 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 is 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
  • nucleic acid derivative is a compound represented by the general formula (A).
  • R is an OH group or an adjacent nucleotide, and R is hydrogen or OH
  • R is OH group or adjacent nucleotide
  • B is adenine, guanine
  • R is a halogen atom or a sulfonate residue
  • R is an alkylsulfo group having 1 to 6 carbon atoms or an alkylthio group having 1 to 6 carbon atoms.
  • R is H or an alkyl group having 1 to 6 carbon atoms
  • n is an integer of 1-6.
  • n is an integer of 1-6.
  • R is hydrogen), an unsubstituted or substituted alkyl group having 1 to 6 carbon atoms
  • R is Hydrogen
  • R ′ is H, an unsubstituted or substituted alkyl carboalkyl, alkyl carboloxy or aryl carborooxy group having 2 to 6 carbon atoms.
  • X is an alkanesulfol group or a benzenesulfol group.
  • N is an integer of 1 to 6
  • X and y are independently 0 to: an integer of L00
  • N is a group represented by the following general formula (B).
  • R is an OH group or an adjacent nucleotide, and R is hydrogen or OH
  • R is OH group or adjacent nucleotide
  • B is adenine, guanine
  • 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.
  • 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,
  • flavin mononucleotide sugar phosphate, sugar nucleotide, coenzyme A, and phospholipid.
  • R is a halogen atom or a sulfonate residue
  • R is an alkylsulfo group having 1 to 6 carbon atoms or an alkylthio group having 1 to 6 carbon atoms.
  • R is H or an alkyl group having 1 to 6 carbon atoms
  • n is an integer of 1-6.
  • 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.
  • 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,
  • 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.
  • the separated conjugate is treated with an alkaline solution to dissociate the conjugate,
  • photoreactive nucleic acid derivatives and nucleotide derivatives can be provided.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • N is a group represented by the following general formula (B).
  • R is an OH group or an adjacent nucleotide.
  • Applicable N is nucleic acid
  • R is an OH group when at the end of the derivative, otherwise R is adjacent
  • R is hydrogen or an OH group. When R is hydrogen, the nucleus
  • the acid derivative is DNA, and when R is an OH group, the nucleic acid derivative is RNA. 1 bottle
  • Nucleic acid derivatives Nucleotide in which R is hydrogen (N) and R is OH
  • Chid (N) can be mixed.
  • R is an OH group or adjacent nucleotide.
  • R is an OH group when the corresponding N is the end of the nucleic acid derivative, otherwise
  • R is the adjacent nucleotide (N).
  • B is adenine, guanine, cytosine, ura
  • 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.
  • 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.
  • 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.
  • 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.
  • the ferrulediaziridine compound can be, for example, a compound represented by the general formula (C).
  • R represents a halogen atom (for example, Cl, Br, 1) or a sulfonate ester residue.
  • 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
  • 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;
  • 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 phenyldiaziridine 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 a phenyldiaziridine compound of the formula (2) or the formula (7).
  • the phenyldiaziridine 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) 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].
  • the ferrulediaziridine compound represented by the general formula (C) can be, for example, a compound represented by the following formula (3).
  • Non-Patent Document 6 M. Kaneda, Y. Sadakane, Y. Hatanaka,
  • the phenyldiaziridine compound represented by the general formula (C) can be, for example, compounds represented by the following formulas (4) to (10).
  • 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.
  • 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.
  • 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.
  • 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.
  • an appropriate functional group is required. Synthetic complexity at that stage was a problem.
  • (12) has been reported as a low-yield synthesis method through 7 steps [JM Delfino, SL Schreiber, and FM Richards, J. Am. Chem.
  • CI CHOCH was found to be in trifluoromethanesulfonic acid (TfOH).
  • Lewis acids are A1C1, ZnCl, SbCl, SbCl,
  • 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.
  • 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.
  • 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.
  • N-methylmorpholine, triethylamine and the like can be used in place of diisopropylethylamine.
  • 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.
  • 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.
  • 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.
  • 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).
  • a disulfide bond is formed to produce a phenyldiaziridine-added nucleic acid derivative (D2).
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • R and R are independently nicotinamide adenine nucleotide and its
  • Phosphorus oxide and flavin mononucleotide, sugar phosphate, sugar nucleotide, coenzyme A, and phospholipid strength are selected.
  • 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.
  • sugar nucleotides include ADP-sugar, CDP-sugar, GDP-sugar, UDP-sugar, and TDP-sugar.
  • 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.
  • coenzyme A examples include acetyl acetylenzyme A.
  • the phospholipid include glyceguchi phospholipid, phosphatidylcholine (lecithin), phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol (cardiolipin), and sphingophospholipid.
  • 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.
  • the present invention includes a method for analyzing a protein by electrical mobility shift assay.
  • 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.
  • the analysis method includes the following steps.
  • 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
  • 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. .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention encompasses a method for preparing a protein.
  • 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.
  • the method for preparing a protein includes the following steps.
  • Steps (d) and (e) can be carried out in the same manner as steps (a) and (b).
  • 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.
  • the conjugate is separated with beads fixed with avidin as a complex with the protein. Capture and implement.
  • 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.
  • 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 shows the chemical state of the dissociation of the conjugate.
  • (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).
  • 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.
  • 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. .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Piotin is originally bound to photoreactive DNA.
  • 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.
  • Lane “ ⁇ ” 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.
  • 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.
  • FIG. 8 shows a schematic diagram of the example below.
  • 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.
  • Detection Example 2 does not perform photoreaction and does not perform the fixed ⁇ 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.
  • 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.
  • 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.
  • 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
  • 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”.
  • Diazirine was introduced into the phosphorothioates of GTP- ⁇ -S and ADP- ⁇ -S shown below.
  • 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.
  • SDS-PAGE (13% polyacrylamide gel
  • streptavidin-HRP peroxidase The results are shown in FIG.
  • the reaction scheme is shown below.
  • $ 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.
  • 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.
  • 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- ⁇ -S by HPLC.
  • FIG. 11-2 Analysis of introduction reaction of diazirine into ADP- ⁇ -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. 14 Reaction scheme in Example 9.

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Abstract

Le problème à résoudre dans le cadre de cette invention concerne des dérivés d’acide nucléique ou de nucléotides photoréactifs, un procédé d’analyse complet de protéine avec les dérivés, etc. La solution proposée consiste en des dérivés d’acide nucléique présentant des fragments de phényldiaziridine, conformément à la formule générale (D) : [Formule chimique 1] (D) ; un procédé de production des dérivés consistant à faire réagir un dérivé d’acide nucléique, au moins un des groupes phosphate étant un groupe thiophosphate avec un composé phényldiaziridine ; des dérivés de nucléotides présentant des fragment de phényldiaziridine ; leur procédé de production consistant à faire réagir un dérivé de nucléotide représenté par la formule générale (E) ou (F) avec un composé de phényldiaziridine présentant un groupe réagissant avec le groupe thiol du dérivé représenté par la formule générale (E) ou (F) : [Formule chimique 11] (E) et (F) et un procédé d’analyse des protéines par EMSA avec les dérivés d’acide nucléique ou de nucléotide ci-dessus présentant des fragments de phényldiaziridine.
PCT/JP2006/309229 2005-05-09 2006-05-08 Derives d’acide nucleique presentant des fragments de phenyldiaziridine et leur procede de production, derives de nucleotides presentant des fragments de phenyldiaziridine et leur procede de production, procede d’analyse de proteine et procede de preparation de proteine WO2006120992A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008245591A (ja) * 2007-03-30 2008-10-16 Institute Of Physical & Chemical Research 光反応性官能基を有する非天然型アミノ酸組み込みタンパク質の合成方法
JP2017530973A (ja) * 2014-09-23 2017-10-19 プロメラス， エルエルシー 光架橋剤としてのジアジリン化合物およびそれを含む光現像性組成物

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KANEDA M. ET AL.: "A Novel Approach for Affinity-Based Screening of Target Specific Ligands: Application of Photoreactive D-Glyceraldehyde-3-phosphate Dehydrogenase", BIOCONJUGATE CHEMISTRY, vol. 14, no. 5, 2003, pages 849 - 852, XP003003639 *
RESEK J.F. ET AL.: "A New Photo-Cross-Linking Reagent for the Study of Protein-Protein Interactions", JOURNAL OF ORGANIC CHEMISTRY, vol. 58, 1993, pages 7598 - 7601, XP002029659 *
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YAMAGUCHI T. ET AL.: "Synthesis and utilization of a photolabile oligodeoxyribonucleotide probe bearing an aryl(trifluoromethyl) diazirine moiety", NUCLEIC ACIDS SYMPOSIUM SERIES, no. 35, 1996, pages 237 - 238, XP008072404 *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008245591A (ja) * 2007-03-30 2008-10-16 Institute Of Physical & Chemical Research 光反応性官能基を有する非天然型アミノ酸組み込みタンパク質の合成方法
JP2017530973A (ja) * 2014-09-23 2017-10-19 プロメラス， エルエルシー 光架橋剤としてのジアジリン化合物およびそれを含む光現像性組成物

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