A kind of specific chemical labeling compound of 5-formyl uracil, labeling method and application

An aldehyde uracil and chemical labeling technology, applied in the field of chemical labeling and detection of epigenetically modified bases, can solve problems such as low 5fU content

Active Publication Date: 2019-07-09
WUHAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] It is precisely because of the relatively small content of 5fU that there is currently no technology for sequencing 5fU in the whole genome

Method used

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  • A kind of specific chemical labeling compound of 5-formyl uracil, labeling method and application
  • A kind of specific chemical labeling compound of 5-formyl uracil, labeling method and application
  • A kind of specific chemical labeling compound of 5-formyl uracil, labeling method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] [Example 1] Synthesis process and characterization of compound 1

[0037] Take 3,4-diaminobenzoic acid (3g, 19.7mmol) and ethyl cyanoacetate (7mL, 65.8mmol) in a 100mL round bottom flask, and stir at 180°C for 1h. After the reaction was completed, the reaction system was poured into 100 mL of diethyl ether to precipitate a solid, which was filtered under reduced pressure. The solid was chromatographed on a silica gel column and eluted with dichloromethane: methanol: acetic acid = 100:1:0.1 to obtain a white solid compound A ( 1 g, 25%). 1 H NMR (400MHz, DMSO-d 6 )δ12.92(s,1H),8.15(s,1H),7.83(d,J=8.4Hz,1H),7.61(d,J=7.7Hz,1H),4.46(s,2H).HRMS( ESI+)C 10 h 8 N 3 o 2 + [M+H] + calculated 202.06110, found 202.06082.

[0038] Concrete reaction route is as follows:

[0039]

[0040] Take compound A (290mg, 1.4mmol), 3-azidopropylamine (720mg, 7.2mmol) and HATU (1.1g, 2.9mmol) in a 25mL round bottom flask, add 10mL N,N-dimethylformamide and 5 drops of triethylamine w...

Embodiment 2

[0043] [Example 2] Compound 1 and 5fU monomer reaction route and characterization

[0044] Take 5-formyluracil (30mg, 0.12mmol) and compound 1 (33mg, 0.12mmol) in a 25mL round bottom flask, add 10mL of methanol and 5 drops of acetic acid, and stir the reaction at 50°C for 15h. After the reaction, the reaction system was evaporated to dryness under reduced pressure, and the solid was eluted by silica gel column chromatography with dichloromethane:methanol=50:1 to 15:1 to obtain compound B (36 mg, 60%) as a yellow solid. 1 H NMR (400MHz, DMSO-d 6 )δ13.39(s,1H),11.98(s,1H),8.95(s,1H),8.57(s,1H),8.36–7.90(m,2H),7.67(dd,J=78.5,12.8Hz ,2H),6.20(t,J=6.6Hz,1H),5.35(s,1H),4.99(s,1H),4.35–4.25(m,1H),3.90(dd,J=6.8,4.1Hz, 1H), 3.63(d, J=4.0Hz, 2H), 3.52–3.40(m, 4H), 2.37–2.11(m, 2H), 1.90–1.72(m, 2H). 13 C NMR (100MHz, DMSO-d 6 )δ167.04,162.12,149.76,143.38,142.24,138.00,135.09,129.33,123.61,122.04,118.71,116.68,111.55,107.86,100.39,88.68,86.20,71.21,61.86,49.06,40.78,37.22,28.94.HRMS...

Embodiment 3

[0047] [Example 3] Marking of mouse genomic DNA

[0048] The extracted genomic DNA (gDNA) was disrupted by an ultrasonic instrument and dissolved in ultrapure water, and the gDNA was quantified by a Qubit Fluorometer quantifier. The labeling of genomic DNA is mainly divided into two steps, such as figure 1 As shown, the first step: Add 20 μL of gDNA (1 μg / μL), 5 μL of sodium acetate buffer (pH 5.0), 5 μL of compound 1 (250 mM dissolved in DMSO) and 20 μL of ultrapure water to a 1.5 mL EP tube , shake and centrifuge, put it into a shaking reactor, and react at 37°C for 12 hours. After the reaction, excess compound 1 was removed with a gel column. Step 2: Add 5 μL of DBCO-S-S-PEG to the product purified in the first step 3 -biotin, in the same way, after shaking and mixing, put it in a shaker at 37°C for 2 hours, and then use a gel column to remove excess small molecule compounds. The final purified mixture was made up to a volume of 100 μL with ultrapure water.

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Abstract

The invention discloses compounds for specifically and chemically marking 5-aldehyde uracil and a marking method and application. A compound 1 with an azide group can be selectively reacted with an aldehyde group of 5-aldehyde uracil, and then alkynyl-carrying biotin DBCO-S-S-PEG3-biotin is added to generate an click reaction with azide without a catalyst. By using the 5-ldehyde uracil specific chemical marking method disclosed by the invention, specific enriching of nucleic acid samples containing 5-aldehyde uracil can be realized, and the sequence distribution information of the 5-aldehyde uracil in nucleic acid molecules can be analyzed. The invention provides an effective research method for the field of epigenetics and nucleic acid chemicobiology.

Description

technical field [0001] The present invention relates to chemical labeling and detection methods of epigenetic modified bases, in particular to a compound 1 and a method for using the compound to specifically chemically label 5-formyl uracil, and its application in labeling, detection, sequencing, etc. application. Background technique [0002] In addition to the four natural bases, there are also a large number of modified bases in nature. Among them, 5-formyluracil (hereinafter referred to as 5fU), an important product of DNA oxidative damage, is a naturally occurring modified base with a small content in the genome. And because it will produce mismatches in the process of DNA replication, such as the pairing of 5fU and guanine, which will cause gene mutations, and it is also one of the important factors that induce various diseases. In the process of active demethylation of nucleic acid in organisms, it is accompanied by oxidative damage and excision repair of modified b...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D235/16C07H19/073C07H1/00C12Q1/6869C12Q1/6806
Inventor 周翔王雅芬刘朝兴吴凡
Owner WUHAN UNIV
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