JPWO2019217533A5 - - Google Patents

Description

を有する化合物に関し、式中、Yは、アルキン、アジド、歪みアルキン、ジエン、ジエノフィル、アルコキシアミン、カルボニル、ホスフィン、ヒドラジド、チオール、およびアルケンより選択されるクリックケミストリー部分であり得；かつ、Xはリンカーであり得る。ある局面において、クリックケミストリー部分(Y)はアジドである。リンカー(X)は、C1、C2、C3、C4、C5、C6、C7、C8、C9、～C10アルキル(1つまたは複数の態様は具体的に除外され得る)またはポリエチレングリコールリンカーであり得る。ある局面において、リンカー(X)はCH₂である。ある局面において、Xは、アルキン、アジド、歪みアルキン、ジエン、ジエノフィル、アルコキシアミン、カルボニル、ホスフィン、ヒドラジド、チオール、およびアルケンより選択されるクリックケミストリー部分であり得；かつ、Yはリンカーであり得る。ある局面において、クリックケミストリー部分(X)はアジドである。リンカー(Y)は、C1、C2、C3、C4、C5、C6、C7、C8、C9、～C10(1つまたは複数の態様は具体的に除外され得る)アルキルまたはポリエチレングリコールリンカーであり得る。ある局面において、リンカー(Y)はCH₂である。
One embodiment is the general formula:

In the formula, Y can be a click chemistry moiety selected from alkynes, azides, strained alkynes, dienes, dienophiles , alkoxyamines, carbonyls, phosphines, hydrazides, thiols, and alkenes; X can be a linker. In one aspect, the click chemistry part (Y) is an azide. The linker (X) can be a C1, C2, C3, C4, C5, C6, C7, C8, C9, ~ C10 alkyl (one or more embodiments may be specifically excluded) or a polyethylene glycol linker. In one aspect, the linker (X) is CH ₂ . In one aspect, X can be a click chemistry moiety selected from alkynes, azides, strained alkynes, dienes, dienophiles , alkoxyamines, carbonyls, phosphines, hydrazides, thiols, and alkenes; and Y is a linker. possible. In one aspect, the click chemistry part (X) is an azide. The linker (Y) can be a C1, C2, C3, C4, C5, C6, C7, C8, C9, ~ C10 (one or more embodiments may be specifically excluded) alkyl or polyethylene glycol linkers. In one aspect, the linker (Y) is CH ₂ .

A. Labeling Reagents, Kethoxal Derivatives As described herein, N ₃ -kethoxal (representing kethoxal derivatives) has been shown to selectively react with guanine in single-stranded DNA and RNA. These reactions are highly efficient under mild standard cell culture conditions and can be applied directly to tissues. Cell-based labeling can be completed within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 minutes (one or more embodiments may be specifically excluded) and this probe It turned out to be a unique probe that has never been seen in the art to date. Its reactivity to DNA enables genome-wide mapping of single-stranded DNA for the first time. The formation of single-stranded DNA on the gene coding region strongly correlates with gene transcription. N ₃ -kethoxal-auxiliary RNA labeling results in RNA secondary structure and RNA-RNA interaction determination. Comparing RNA structures in vitro and in vivo identifies factors that mediate RNA structure dynamics in living cells, such as gene translation and RNA binding proteins. In addition, further applications such as N ₃ -kethoxal photoregulated in situ RNA labeling, exposure RNA mapping, single molecule DNA / RNA imaging, small RNA binding agent screening, etc. by combining N ₃ -kethoxal with other techniques. Any chemical moiety can be attached onto the kethoxal derivative using the methods described herein. Among the specific uses according to some aspects of the invention are click chemistry handles. Click chemistry handles are chemical moieties that provide reactive groups that can participate in click chemistry reactions. Suitable chemical groups for click chemistry and click chemistry reactions are well known to those of skill in the art and are terminal alkynes, azides, strained alkynes, dienes, dienophiles , alkoxyamines, carbonyls, phosphines, hydrazides, thiols, and alkenes. , But not limited to these. For example, in some embodiments, azides and alkynes are used in click chemistry reactions. In certain aspects, the "click chemistry compatibility" compound or click chemistry handle comprises a terminal azide functional group.

ある局面において、Yは、保護チオール、アルケン(TCOを含む)およびテトラジン逆要請型Diels-Alder、テトラゾールフォトクリック反応、ビニルチオエーテルアルキン、アジド、歪みアルキン、ジエン、ジエノフィル、アルコキシアミン、カルボニル、ホスフィン、ヒドラジド、チオール、およびアルケンより選択されるクリックケミストリー適合性反応基であり；かつ、Xは、リンカー(C1、C2、C3、C4、C4、C5、C6、C7、C8、C9～C10アルキルまたはそれ以上(1つまたは複数の態様は具体的に除外され得る)、ポリエチレングリコールなど)である。他の局面において、Xは、保護チオール、アルケン(TCOを含む)およびテトラジン逆要請型Diels-Alder、テトラゾールフォトクリック反応、ビニルチオエーテルアルキン、アジド、歪みアルキン、ジエン、ジエノフィル、アルコキシアミン、カルボニル、ホスフィン、ヒドラジド、チオール、およびアルケンより選択されるクリックケミストリー適合性反応基であり；かつ、Yは、リンカー((C1、C2、C3、C4、C4、C5、C6、C7、C8、C9～C10アルキルまたはそれ以上(1つまたは複数の態様は具体的に除外され得る)、ポリエチレングリコールなど)である。

In one aspect, Y is a protected thiol, an alkene (including TCO) and a tetradine reverse-requested Diels-Alder, a tetrazole photoclick reaction, a vinylthioether alcoholine, an azide, a strained alkene, a diene, a dienophile , an alkoxyamine, a carbonyl, It is a click chemistry compatible reactive group selected from phosphines, hydrazides, thiols, and alkenes; and X is a linker (C1, C2, C3, C4, C4, C5, C6, C7, C8, C9 to C10 alkyl. Or more (one or more embodiments may be specifically excluded), such as polyethylene glycol). In other aspects, X is a protected thiol, an alkene (including TCO) and a tetradine reverse-requested Diels-Alder, a tetrazole photoclick reaction, a vinylthioether alkin, an azide, a strained alkene, a diene, a dienophile , an alkoxyamine, a carbonyl. , A click chemistry compatible reactive group selected from phosphines, hydrazides, thiols, and alkenes; and Y is a linker ((C1, C2, C3, C4, C4, C5, C6, C7, C8, C9 ~). C10 alkyl or higher (one or more embodiments may be specifically excluded), polyethylene glycol, etc.).

Kethoxal derivatives can be modified to include protein cross-linking agents such as diazirine or benzophenone. The current CLIP-seq protocol involves UV initiation cross-linking of amino acid side chains, including aromatic side chains, to RNA bases. These cross-linking reactions suffer from extremely low efficiency. The ketoxal bifunctional molecules described herein are composed of ketoxal, which allows for efficient RNA labeling, and an efficient protein cross-linking agent, such as diazirine or benzophenone. Diazirine and benzophenone form radicals upon UV irradiation. The cross-linking agent is capable of capturing any nearby primary carbon in the vicinity to form a covalent bond, thus making it a suitable reagent for protein cross-linking. Binding of diazirine or benzophenone to kethoxal derivatives allows stabilization of their weak protein interactions. In this way, pools of RNA that interact with or are close to the target protein can be fished via pull-down with antibodies specific for the protein of interest.

Examples of protein cross-linking agents include dysuccinimidyl glutarate, dysuccinimidyl suberate, dysuccinimidyl tartrate, dimethyl adipimide, dimethyl pimmelimide, dimethyl suberimide, 1,5-difluoro-2,4-dinitrobenzene. , N-maleimide propionic acid hydrazide, 3- (2-pyridyldithio) propionylhydrazide, bismaleimideethane, diazilin , iodoacetate succinimidyl, N-maleimide acetoxysuccinimid ester, 3- (2-pyridyldithio) propionate succinimidyl, And benzophenone, but not limited to these.

Claims (14)

formula:

It is a compound having
In the formula, Y is the click chemistry moiety selected from alkynes, azides, strained alkynes, dienes, dienophils , alkoxyamines, carbonyls, phosphines, hydrazides, thiols, and alkenes;
X is a C1-C10 alkyl or polyethylene glycol linker,
The compound. The compound according to claim 1, wherein Y is an azide. The compound according to claim 1 or 2 , wherein X is CH ₂ . formula:

Compounds with. A method for labeling guanine bases,
It comprises contacting the labeled guanine with the compound according to any one of claims 1 to 4 to form a reaction mixture, and incubating the reaction mixture at 30-40 ° C. for at least 5 minutes. The method. The method of claim 5 , wherein the polynucleotide contains guanine. A method for labeling single-stranded nucleic acids in cells.
(i) A step of contacting the target cell with the compound according to claim 1 to form a treated cell containing a nucleic acid having a kethoxal derivative-labeled guanine base ;
(ii) The step of contacting the treated cell with at least two click chemistry reactive moieties and a cross-linked moiety containing a tag, wherein the cross-linked moiety cross-links two adjacent ketoxal derivative labeled guanines to form a cross-linked nucleic acid. Forming, steps; and
(iii) The method comprising isolating a crosslinked nucleic acid using a reagent that is fragmented and has an affinity for the tag. The method according to claim 7 , wherein the tag is biotin and the reagent having an affinity for the tag is streptavidin . The method of claim 7 or 8 , wherein the click chemistry reactive moiety is a dibenzocyclooctyne moiety. A method for RNA secondary structure mapping and RNA G-quadruplex prediction of the entire transcriptome in vivo.
(a) In vivo labeling of nucleic acid with a ketoxal click chemistry derivative to produce labeled nucleic acid ;
(b) A step of biotinlating a labeled nucleic acid to produce a labeled and biotinylated nucleic acid ;
(c) Fragmentation of labeled and biotinylated nucleic acid to produce fragmented nucleic acid ;
(d) Step of synthesizing complementary DNA (cDNA) from fragmented nucleic acid ;
(e) Step of isolating c DNA;
(f) Step of separating cDNA based on size;
(g) The step of cyclizing the separated cDNA to form a cyclized cDNA library ; and
(h) The method comprising the step of amplifying a cyclized cDNA library. 10. The method of claim 10 , wherein the cyclization of the separated cDNA comprises ligation of the cDNA with a single-stranded DNA ligase. A method for analyzing R NA / protein interactions,
(a) The step of contacting a protein target in close proximity to RNA with a ketoxal derivative bound to an activating protein crosslinkable moiety to form a target / kethoxal derivative mixture;
(b) The step of exposing the target / kethoxal derivative mixture to the activator to activate the protein crosslinkable moiety to form a crosslinked target / kethoxal derivative complex;
(c) Isolation of the crosslinked target / kethoxal derivative complex using an affinity substance that binds to the protein target; and
(d) The method comprising identifying an RNA molecule isolated with a crosslinked target / kethoxal derivative complex. The activable crosslinkable moiety is dysuccinimidyl glutarate, dysuccinimidyl suberate, dysuccin mityl tartrate, dimethyl adipimide, dimethyl pimmelimide, dimethyl suberimide, 1,5-difluoro-2, 4-Dinitrobenzene, N-maleimide propionic acid hydrazide, 3- (2-pyridyldithio) propionylhydrazide, bismaleimideethane, diazilin , iodoacetate succinimidyl, N-maleimide acetoxysucciniimide ester, 3- (2-pyridyldithio) 12. The method of claim 12 , which is succinimidyl propionate or benzophenone. A method for synthesizing azido-kethoxal (N ₃ -kethoxal),
(a) (i) Mixing sodium hydride with 2-azidoethanol in tetrahydrofuran (THF) to form the first intermediate mixture,
(ii) Add ethyl 2-bromopropionate to the first intermediate mixture to form the first reaction mixture,
(iii) Incubating the first reaction mixture in a nitrogen (N ₂ ) atmosphere at room temperature to form 2- (2-azidoethoxy) propanoic acid,
(iv) Quenching the reaction with water,
(v) Add 2- (2-azidoethoxy) propionic acid to an aqueous LiOH solution and incubate at room temperature, and
(vi) A step of producing a 2- (2-azidoethoxy) propionic acid intermediate by washing, isolating and drying 2- (2-azidoethoxy) propionic acid with anhydrous Na ₂ SO ₄ ;
(b) Dissolving (i) 2- (2-azidoethoxy) propanoic acid in anhydrous CH ₂ Cl ₂ and dimethylformamide (DMF),
(ii) Add oxalyl chloride and stir at room temperature,
(iii) Removing DMF and excess oxalyl chloride to form a residue,
(iv) Dissolving the residue in anhydrous CH ₃ CN and (trimethylsilyl) diazomethane,
(v) Add diethyl ether to form a second reaction mixture,
(vi) Stir the second reaction mixture at 0 ° C. for at least 8 hours ,
(vii) Intermediate 3- (2-azidoethoxy) -1-diazopentan-2-one by evaporating the solvent to isolate 3- (2-azidoethoxy) -1-diazopentan-2-one. The process of creating a body; as well
(c) (i) Add 3- (2-azidoethoxy) -1-diazopentan-2-one to dimethyldioxirane (DMD-acetone) in acetone to form a third reaction mixture,
(ii) Stir the third reaction mixture at room temperature to form N ₃ -ketoxal or 3- (2-azidoethoxy) -1,1-dihydroxybutane-2-one, and
(iii) By isolating N ₃ -kethoxal or 3- (2-azidoethoxy) -1,1-dihydroxybutane-2-one, N ₃ -kethoxal or 3- (2-azidoethoxy) -1, The method comprising the step of producing 1-dihydroxybutane-2-one.