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
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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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