Method for preparation and purification of four stereoisomers of benzopyrene-DNA adduct

Abstract

The invention discloses a method for preparation and purification of four stereoisomers of a benzopyrene-DNA adduct. An in-vitro synthesis mode is adopted to synthesize the four stereoisomers of the benzopyrene-DNA adduct which is benzopyrene-7,8-dihydrodiol-9,10-epoxide-deoxyguanosine adduct (anti-BPDE-N<2>-dG), and through optimization of the in-vitro reaction conditions, the yield of the synthesized four stereoisomers of anti-BPDE-N<2>-dG is improved by more than 2 times when the method is compared with an existing synthesis method. A pi-hole effect is applied to chromatographic separationand purification of the stereoisomers for the first time, and the four stereoisomers can be separated completely in a short time through optimization of the chromatographic conditions, so that the chromatographic purification time is shortened greatly, and the purification efficiency is improved.

Description

technical field

[0001] The method for preparing and purifying four stereoisomers of benzopyrene DNA adducts of the invention belongs to the technical field of DNA adduct synthesis of polycyclic aromatic hydrocarbons. Background technique

[0002] Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic pollutants that widely exist in the environment. They mainly come from the incomplete combustion of organic compounds such as coal and petroleum. will also generate. Among them, benzo[a]pyrene (B[a]p) is the most typical polycyclic aromatic hydrocarbon, which has strong teratogenic, carcinogenic and mutagenic properties. After entering the human body, it undergoes a series of metabolic activation to generate 7 ,8 diol-9,10 epoxy benzopyrene (benzo[a] pyrene-7,8-dihydrodiol-9,10-epoxide, BPDE). This metabolite has two pairs of enantiomers (±)anti-BPDE and (±)syn-BPDE (diastereoisomers between anti-BPDE and syn-BPDE), a large number of studies have shown that ...

Examples

Embodiment 1

[0031] 1. Synthesis of anti-BPDE-N 2 -dG

[0032] Weigh 0.1 mg (±)anti-BPDE and dissolve in 100 μL of tetrahydrofuran: triethylamine=19:1 solution; weigh 4.8 mg of 2'deoxyguanosine (dG) and dissolve in 1.2 mL 50 mmol / L Tris -HCl, pH=7.5 buffer solution, mix the two, avoid light, and react at room temperature at 23.6°C for 48 hours.

[0033] 2. Preliminary purification by solid phase extraction

[0034] To extract the target product anti-BPDE-N from the reaction solution 2 -dG, requires solid phase extraction for preliminary purification and enrichment, and removes the reaction base solution. Weigh 100 mg Si-C 18 Adsorbent, methanol was activated and loaded into the solid-phase extraction column, 5 mL of water was replaced by methanol, the reaction solution was passed through the activated solid-phase extraction column, and then 2 mL of ultrapure water, 1 mL of 10% methanol aqueous solution were used successively, Rinse with 1 mL of 30% methanol aqueous solution, drain the...

Embodiment 2

[0058] 1. Synthesis of anti-BPDE-N 2 -dG

[0059] Weigh 0.2 mg (±) anti-BPDE and dissolve it in 100 μL of tetrahydrofuran: triethylamine = 17:1 solution to obtain A solution, the concentration of BPDE in A solution is: 2 g / L; weigh 8.9 mg of 2'deoxyguanosine (dG) was dissolved in 1.2 mL of 50 mmol / LTris-HCl pH=7 buffer solution to obtain solution B. In solution B, the concentration of 2'deoxyguanosine was 7.4 g / L; Mix with solution B so that the ratio of BPDE to 2'deoxyguanosine is 1:50, mix well, keep away from light, and react at room temperature at 20°C for 50 hours.

[0060] 2. Initial purification

[0061] Weigh Si-C 18 The adsorbent is activated by methanol and loaded into the solid phase extraction column. The crude product solution is passed through the activated solid phase extraction column, and then rinsed with ultrapure water and methanol aqueous solution of different concentrations, and finally the column is eluted with methanol. Collect the eluent and concent...

Embodiment 3

[0066] 1. Synthesis of anti-BPDE-N 2 -dG

[0067] Weigh 0.05 mg (±) anti-BPDE and dissolve it in 100 μL of tetrahydrofuran: triethylamine = 23:1 solution to obtain A solution, the concentration of BPDE in A solution is: 0.5g / L; weigh 2.6 mg of 2'deoxyguanosine (dG) was dissolved in 1.2 mL 50 mmol / LTris-HCl pH=9 buffer solution to obtain solution B. In solution B, the concentration of 2'deoxyguanosine was 2.2 g / L, and solution A Mix with solution B so that the ratio of anti-BPDE to 2'deoxyguanosine is 1:60, mix well, keep away from light, and react at room temperature at 28°C for 40 hours.

[0068] 2. Initial purification

[0069] Weigh Si-C 18 The adsorbent is activated by methanol and loaded into the solid phase extraction column. The crude product solution is passed through the activated solid phase extraction column, and then rinsed with ultrapure water and methanol aqueous solution of different concentrations, and finally the column is eluted with methanol. Collect the...

technical field

[0001] The method for preparing and purifying four stereoisomers of benzopyrene DNA adducts of the invention belongs to the technical field of DNA adduct synthesis of polycyclic aromatic hydrocarbons. Background technique

[0002] Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic pollutants that widely exist in the environment. They mainly come from the incomplete combustion of organic compounds such as coal and petroleum. will also generate. Among them, benzo[a]pyrene (B[a]p) is the most typical polycyclic aromatic hydrocarbon, which has strong teratogenic, carcinogenic and mutagenic properties. After entering the human body, it undergoes a series of metabolic activation to generate 7 ,8 diol-9,10 epoxy benzopyrene (benzo[a] pyrene-7,8-dihydrodiol-9,10-epoxide, BPDE). This metabolite has two pairs of enantiomers (±)anti-BPDE and (±)syn-BPDE (diastereoisomers between anti-BPDE and syn-BPDE), a large number of studies have shown that ...

Examples

Embodiment 1

[0031] 1. Synthesis of anti-BPDE-N 2 -dG

[0032] Weigh 0.1 mg (±)anti-BPDE and dissolve in 100 μL of tetrahydrofuran: triethylamine=19:1 solution; weigh 4.8 mg of 2'deoxyguanosine (dG) and dissolve in 1.2 mL 50 mmol / L Tris -HCl, pH=7.5 buffer solution, mix the two, avoid light, and react at room temperature at 23.6°C for 48 hours.

[0033] 2. Preliminary purification by solid phase extraction

[0034] To extract the target product anti-BPDE-N from the reaction solution 2 -dG, requires solid phase extraction for preliminary purification and enrichment, and removes the reaction base solution. Weigh 100 mg Si-C 18 Adsorbent, methanol was activated and loaded into the solid-phase extraction column, 5 mL of water was replaced by methanol, the reaction solution was passed through the activated solid-phase extraction column, and then 2 mL of ultrapure water, 1 mL of 10% methanol aqueous solution were used successively, Rinse with 1 mL of 30% methanol aqueous solution, drain the...

Embodiment 2

[0058] 1. Synthesis of anti-BPDE-N 2 -dG

[0059] Weigh 0.2 mg (±) anti-BPDE and dissolve it in 100 μL of tetrahydrofuran: triethylamine = 17:1 solution to obtain A solution, the concentration of BPDE in A solution is: 2 g / L; weigh 8.9 mg of 2'deoxyguanosine (dG) was dissolved in 1.2 mL of 50 mmol / LTris-HCl pH=7 buffer solution to obtain solution B. In solution B, the concentration of 2'deoxyguanosine was 7.4 g / L; Mix with solution B so that the ratio of BPDE to 2'deoxyguanosine is 1:50, mix well, keep away from light, and react at room temperature at 20°C for 50 hours.

[0060] 2. Initial purification

[0061] Weigh Si-C 18 The adsorbent is activated by methanol and loaded into the solid phase extraction column. The crude product solution is passed through the activated solid phase extraction column, and then rinsed with ultrapure water and methanol aqueous solution of different concentrations, and finally the column is eluted with methanol. Collect the eluent and concent...

Embodiment 3

[0066] 1. Synthesis of anti-BPDE-N 2 -dG

[0067] Weigh 0.05 mg (±) anti-BPDE and dissolve it in 100 μL of tetrahydrofuran: triethylamine = 23:1 solution to obtain A solution, the concentration of BPDE in A solution is: 0.5g / L; weigh 2.6 mg of 2'deoxyguanosine (dG) was dissolved in 1.2 mL 50 mmol / LTris-HCl pH=9 buffer solution to obtain solution B. In solution B, the concentration of 2'deoxyguanosine was 2.2 g / L, and solution A Mix with solution B so that the ratio of anti-BPDE to 2'deoxyguanosine is 1:60, mix well, keep away from light, and react at room temperature at 28°C for 40 hours.

[0068] 2. Initial purification

[0069] Weigh Si-C 18 The adsorbent is activated by methanol and loaded into the solid phase extraction column. The crude product solution is passed through the activated solid phase extraction column, and then rinsed with ultrapure water and methanol aqueous solution of different concentrations, and finally the column is eluted with methanol. Collect the...

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