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Fluorescence detection method of five-position aldehyde-group deoxidizing uridine

An aldehyde-based deoxyuridine and detection reagent technology, applied in fluorescence/phosphorescence, material excitation analysis, etc., can solve the problems of large required amount, complex cost, weak selectivity, etc., and achieve the effect of strong selectivity

Inactive Publication Date: 2012-08-01
WUHAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] There have been some achievements in the field of DNA damage detection, but there are few reports on the detection of oxidative damage to deoxythymidine
Some existing reports include biological and chemical detection, but biological detection has its disadvantages such as complexity and high cost. Some chemical methods cannot be simple and quick, and some are not strong in fluorescence contrast and selectivity. Or the small molecule probes used in the detection are not easy to obtain or the required amount is relatively large

Method used

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  • Fluorescence detection method of five-position aldehyde-group deoxidizing uridine
  • Fluorescence detection method of five-position aldehyde-group deoxidizing uridine
  • Fluorescence detection method of five-position aldehyde-group deoxidizing uridine

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Experimental program
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Effect test

Embodiment 1

[0026] Example 1: 4-methoxy o-phenylenediamine was used to react with deoxycytidine, five-aldeoxycytidine and five-aldeoxyuridine respectively and measure the fluorescence spectrum thereof.

[0027] 4-Methoxy-o-phenylenediamine, penta-aldeoxyuridine and dithiothreitol were prepared as 10 mM solutions in DMSO (dimethyl sulfoxide). Then dilute 10mM 4-methoxy-o-phenylenediamine to 1mM solution for later use, and prepare acetic acid buffer solution with pH=4.5 with ammonium acetate and acetic acid at a concentration of 1M.

[0028] Take 8 μL of 1 mM 4-methoxy-o-phenylenediamine solution, 4 μL of 10 mM 10 mM dithiothreitol solution, 4 μL of 10 mM penta-aldehyde deoxyuridine solution, 4 μL of 1M acetate buffer, and add 380 μL of high-purity water to prepare into 400 μL mixed solution and detect its fluorescence spectrum after reacting for 3 hours, a new peak appeared at 475nm, compared with Figure three The peak of compound A in ? confirms the occurrence of the reaction.

[0029]...

Embodiment 2

[0034] Embodiment 2: Use o-phenylenediamine to react with deoxycytidine, five-formyl deoxycytidine and five-formyl deoxyuridine respectively and measure its fluorescence spectrum

[0035] O-phenylenediamine, penta-aldeoxyuridine and dithiothreitol were prepared into 10 mM solutions in DMSO (dimethyl sulfoxide). Then dilute the 10mM o-phenylenediamine into 1mM solution for later use, prepare the acetate buffer solution with pH=4.5 with ammonium acetate and acetic acid, the concentration is 1M.

[0036] Take 8 μL of 1 mM o-phenylenediamine solution, 4 μL of 10 mM 10 mM dithiothreitol solution, 4 μL of 10 mM penta-aldehyde deoxyuridine solution, 4 μL of 1M acetate buffer, add 380 μL of high-purity water to prepare a 400 μL mixture and mix Detect its fluorescence spectrum after reacting for 3 hours, new peak appears at 430nm place, contrast Figure four The peak of compound B in ? confirms the occurrence of the reaction.

[0037] O-phenylenediamine, penta-aldeoxycytidine and dit...

Embodiment 3

[0042] Embodiment 3: the chemical synthesis of compound A

[0043] Dissolve 17mg of 4-methoxy-o-phenylenediamine and 30mg of penta-aldehyde deoxyuridine in 2mL of DMF solvent. After stirring at room temperature for one hour, add 6mg of catalyst scandium trifluoromethanesulfonate and 12μL of oxidant hydrogen peroxide (30%) , and exposed to the air to react. After the reaction was detected by TLC, the solvent was evaporated under reduced pressure, and the column was purified with eluent (chloroform:methanol=20:1) to obtain 29.8mg of compound A, the yield was 68%, and it was brown yellow solid, 1 H NMR (300MHz DMSO-d 6 ) δ 2.213 (s, 2H), 3.596 (s, 2H), 3.744 (s, 3H), 3.856 (s, 1H), 4.269 (s, 1H), 5.061 (s, 1H), 5.331 (s, 1H) , 6.197 (t, J=6.3Hz, 1H), 6.755 (d, J=8.4Hz, 1H), 7.093 (d, J=19.2Hz, 1H), 7.439 (t, J=8.1Hz, 1H), 8.743 (d, J=12Hz, 1H), 11.910 (s, 1H), 12.021 (s, 1H); 13 C NMR (300MHz DMSO-d 6 ) δ 19.222, 55.990, 56.083, 56.701, 62.069, 71.168,71.225,85.834,88.429,88...

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Abstract

The invention relates to a chemical method for detecting how deoxythymidine is oxidized and mutated into five-position aldehyde-group deoxidizing uridine, which includes: using 4-methoxy group o-phenylenediamine or o-phenylenediamine as a detection reagent, using dithiothreitol as an antioxidant reagent, and dissolving the detection reagent and the antioxidant reagent into 10mM of acetic acid buffer liquid with the potential of hydrogen (pH) of 4.5 to form a detection sample; and leading the detection sample to react for 3 hours under aerobic room temperature condition and using a fluorophotometric detector to detect a blank sample and the detection sample, wherein through the detection, users can find out that when the 4-methoxy group o-phenylenediamine is used as the detection reagent, the peak value shifts to 475nm, and when the o-phenylenediamine is used as the detection reagent, the peak value shifts to 430nm. The fluorescence detection method generates compounds capable of generating fluorescence by using the 4-methoxy group o-phenylenediamine or the o-phenylenediamine and 5-aldehyde group deoxyuridine which are easy to achieve and are arranged in acetic acid buffer liquid so that existence of oxidation and mutation of the deoxythymidine can be detected in fluorescence mode.

Description

[0001] technical field [0002] The invention relates to a fluorescence detection method for oxidative mutation of deoxythymidine into five-formyl deoxyuridine, which belongs to the field of biological detection. Background technique [0003] Deoxythymidine in living cells is easily oxidized by reactive oxygen species produced by ultraviolet rays and ionizing radiation to cause damage. The five-aldehyde deoxyuridine is the product of deoxythymidine oxidative damage. Therefore, a simple and sensitive method for detecting five-formyl deoxyuridine has important significance and application value for early diagnosis of diseases and even life sciences. Fluorescent probes have always been a simple and sensitive detection method. If mutations at the nucleic acid level can be observed through simple fluorescence detection, it will be of great significance for the detection of oxidative damage to deoxythymidine. [0004] There have been some achievements in the detection of DNA ...

Claims

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Application Information

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IPC IPC(8): G01N21/64
Inventor 周翔郭璞翁小成
Owner WUHAN UNIV
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