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Hybrid azaguanosine as well as synthesis method and application thereof in DNA sequencing

A technology of guanine nucleoside and guanine nucleotide, which is applied in the fields of chemical synthesis and biochemistry, and can solve the problems of inapplicability to large-scale production, low yield, and high price

Active Publication Date: 2014-05-21
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In patent CN201310489350.4 and patent CN201310489397.0, the price of raw materials required for glycosidation reaction is high, and the yield of reaction is very low, which is not suitable for large-scale production

Method used

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  • Hybrid azaguanosine as well as synthesis method and application thereof in DNA sequencing
  • Hybrid azaguanosine as well as synthesis method and application thereof in DNA sequencing
  • Hybrid azaguanosine as well as synthesis method and application thereof in DNA sequencing

Examples

Experimental program
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Effect test

Embodiment 1

[0076] The synthetic method of embodiment 1,7-deaza-7-iodo-8-aza-2'-deoxyguanosine

[0077] The synthesizing scheme of formula (I1-iodine) compound (i.e. 7-deaza-7-halogen-8-aza-guanosine) in this example is as follows figure 1 As shown, the specific synthetic method comprises the following steps respectively:

[0078] step one,

[0079]

[0080] Dissolve 2-amino-4,6-dihydroxypyrimidine (12.5 g, 100 mmol) in 75 mL POCl 3 and 17.5mL of anhydrous DMF, stirred at room temperature for 10min, under nitrogen protection, refluxed at 105°C for 1.5h, stopped heating, cooled to room temperature, spin out the solvent, dissolved the solid in 2L of ice water, stirred at 50°C for 2h Filter, wash with water and ethyl acetate, and dry in vacuo to obtain 11.0 g of light yellow solid with a yield of 57.6%.

[0081] 1 H NMR (400MHz, DMSO): δ = 10.05 (s, 1H, -CHO), 8.49 (brs, 2H, -NH 2 ).

[0082] Step two,

[0083]

[0084] The formula (Ⅷ) compound (2.4g, 12.5mmol) was added into ...

Embodiment 2、7

[0107] The synthetic method of embodiment 2,7-deaza-7-iodo-8-aza-guanosine

[0108] The synthetic schematic diagram of (I2-iodine) in the present embodiment is as follows figure 2 As shown, the specific synthetic method comprises the following steps respectively:

[0109] Step 1, when compound VI, R 3 for , the specific reaction formula is as follows:

[0110] (Ⅲ2-iodine)

[0111] Add the compound of formula (Ⅳ-iodine) (879 mg, 3.02 mmol) into 3 mL of anhydrous methanol, then add 1.25 mL of 2.85 M potassium hydroxide / methanol solution, stir at room temperature for 1 min, add 3 mL of toluene, spin out the solvent, and dry the solid in vacuum After that, add 3.5mL of anhydrous acetonitrile and 1.5mL of anhydrous DMF mixed solution, nitrogen protection, add 1-chloro 2,3,5-di-p-chlorobenzoyloxy-2-deoxy-D-ribofuranose (1.79 g, 3.06mmol), after stirring at room temperature for 24h, spin out the solvent, column chromatography [V (petroleum ether): V (ethyl acetate) = 5: 1]...

Embodiment 3

[0122] Example 3, 7-deaza-7-iodo-8-aza-2'-deoxyguanosine in the synthesis of dG(8-aza)TP(AP 3 ) used in way

[0123] In this example, dG(8-aza)TP(AP 3 ) Synthesis schematic diagram as image 3 As shown, the specific synthetic method comprises the following steps respectively:

[0124] step one,

[0125]

[0126] Add formula (I 1-iodine) compound (78.6mg, 0.2mmol) in a single-necked bottle, then weigh CuI (9.5mg, 0.5mmol) and Pd (PPh 3 ) 4 (23.1mg, 0.02mmol) was added to the reaction flask, evacuated, nitrogen protected, wrapped in aluminum foil, added 5mL of anhydrous DMF, stirred to dissolve, injected with TEA (40.4mg, 0.4mmol) and trifluoroacetylpropargylamine (90.6mg, 0.6mmol), stirred at 50°C for 12 hours, the reaction was completed, the solvent was spun out, the residue was dissolved in 50mL ethyl acetate, washed with saturated sodium bicarbonate solution and saturated sodium chloride solution successively, dried over anhydrous sodium sulfate, concentrated...

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Abstract

The invention discloses hybrid azaguanosine as well as a synthesis method and an application thereof in DNA sequencing. The synthesis method comprises the steps: removing a protecting group of a compound as shown in formula (III) in the specification under an alkaline condition to obtain a compound as shown in formula (II) in the specification; further demethylating to obtain a compound as shown in formula (I) in the specification, i.e. 7-deaza-7-halogen-8-aza-guanosine, wherein R1 is H or OH, R2 is I, Br or Cl, and R3 is H or a compound shown in the specification. The hybrid azaguanosine disclosed by the invention is a novel reagent for DNA sequencing, which, compared to guanosine failing to substitute nitrogen on 8 sites, is more excellent in base identifying effect and more stable in DNA chain structure. Meanwhile, different from the prior art that 8-site nitrogen-substituted guanosine is complex in synthesis method, low in yield and unsuitable for commercial production, the synthesis method disclosed by the invention is easily available in raw material required, and adopts conventional chemical synthesis reaction; and the method is relatively high in yield, and suitable for wide popularization and application.

Description

technical field [0001] The invention relates to the fields of chemical synthesis and biochemistry, in particular to an azized guanosine, its synthesis method and its application in DNA sequencing. Background technique [0002] The publication of the working draft of the human genome in 2001 marked the arrival of the post-genome era. People's demand for sequencing technology has not decreased but increased, and DNA sequencing technology is still in a period of rapid development. In recent years, the Human Genome Project has promoted the improvement of DNA sequencing efficiency and the reduction of sequencing costs, but the current DNA sequencing methods still have the problems of high cost and time-consuming, which affects the market application of DNA sequencing technology and hinders scientific research. develop. Scientists have been committed to the development of high-throughput and low-cost sequencing technology. Therefore, the third generation of high-throughput sequen...

Claims

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Application Information

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IPC IPC(8): C07H19/167C07H19/173C07H19/20C07H1/00C07H1/04C09K11/06C12Q1/68
CPCY02P20/55
Inventor 沈玉梅魏晓飞龚兵胡蔚姜玉赵小东邵志峰李小卫伍新燕汤道年刘亚智
Owner SHANGHAI JIAO TONG UNIV
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