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Azo linkage unit based fluorescence labeled nucleotide and applications thereof

A ligation unit and fluorescent labeling technology, applied in the field of DNA sequencing, can solve the problems of mild shearing conditions, low efficiency, short read length, etc.

Active Publication Date: 2015-06-24
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the existing cleavable junction units have many disadvantages, such as the shearing conditions are not mild enough, the efficiency is not high, and the read length is too short when used for sequencing.

Method used

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  • Azo linkage unit based fluorescence labeled nucleotide and applications thereof
  • Azo linkage unit based fluorescence labeled nucleotide and applications thereof
  • Azo linkage unit based fluorescence labeled nucleotide and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1. Synthesis of intermediate compound B1

[0073] The synthesis schematic diagram of the intermediate compound B1 of this embodiment is as follows: figure 2 As shown, the specific steps are as follows:

[0074] (1) Synthesis of compound 15: dissolve polyethylene glycol (9g, 60mmol) in 70mL of toluene, then add 10.2mL of 48% HBr aqueous solution, heat to reflux, absorb the emitted gas with sodium bicarbonate, and control the reaction temperature to The reaction was stirred at 115°C for 3 days. After cooling the reaction solution, add saturated sodium bicarbonate aqueous solution to make it neutral, then spin off the solvent, add 30mL water, and use CH 2 Cl 2 Extract (3*60mL). Combine the organic phases, dry with anhydrous sodium sulfate, and spin off the organic solvent to obtain 5.2 g of compound 15 with a yield of 41%. 1 H NMR(400MHz, CDCl 3 ): δppm 3.68(t,2H,J=6.0Hz), 3.58(t,2H,J=4.6Hz), 3.51-3.55(m,4H), 3.44(t,2H,J=4.6Hz), 3.35( t, 2H, J = 6.3 Hz).

[0075] (2)...

Embodiment 2

[0078] Example 2. Synthesis of intermediate compounds A31 and A71

[0079] The synthesis schematic diagram of the intermediate compounds A31 and A71 in this example is as follows: image 3 As shown, the specific steps are as follows:

[0080] (1) Synthesis of compound 2: 4-iodoaniline (4.38g, 20mmol) was dissolved in 30ml ethyl acetate, 2ml of triethylamine was added, benzyl chloroformate (3.4g, 20mmol) was added dropwise under ice water bath, and stirred to The reaction was carried out at room temperature for 4 hours, the aqueous phase of the reaction liquid, the organic phase were dried with anhydrous sodium sulfate, and the solvent was evaporated to dryness to obtain 27.27 g of the product compound with a yield of 97%. 1 H NMR(500MHz, CDCl 3 )δ7.89–7.71(m,4H), 7.40–7.24(m,5H), 7.10(s,1H), 4.65(s,2H);

[0081] (2) Synthesis of compound A11: Add compound 2 (0.7mmol, 247mg) into a single-necked flask, then weigh 9.7mg CuI and 20.3mg Pd(PPh 3 ) 4 Add to the reaction flask, vacuum, ni...

Embodiment 3

[0089] Example 3. Synthesis of intermediate compounds B11 and B12

[0090] The synthesis schematic diagram of the intermediate compounds B11 and B12 in this example is as follows: Figure 4 As shown, the specific steps are as follows:

[0091] (1) Synthesis of compound B11: Dissolve compound phloroglucinol (47mg, 0.372mmol) and potassium carbonate (19mg, 0.136mmol) in 3mL DMF in a 10mL single-neck flask, and dissolve compound B1 (50mg, 0.124mmol) Add 3ml DMF to the reaction flask, and stir at 120°C for 2.5h under nitrogen protection. After cooling to room temperature, 20 mL of ethyl acetate was added, and then washed with water, dried over anhydrous sodium sulfate, and the organic solvent was spun off. The residue was separated by column chromatography to obtain 27 mg of compound B11 with a yield of 59%. 1 H NMR(500MHz, CDCl 3 )δ6.00–5.87(m,3H),4.72(brs,2H),4.59(s,1H), 4.30(s,1H), 3.76(s,1H), 3.66(t,J=8.1Hz,2H ),3.51(s,4H),3.04(t,J=8.1Hz,2H),1.42(s,9H). 13 C NMR(125MHz, CDCl 3 )δ...

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Abstract

The invention provides an azo linkage unit based fluorescence labeled nucleotide and applications thereof. The structural formula of the nucleotide is shown in a formula VI in the specification, wherein fluorescein is selected from one of BODIPY, rhodamine, coumarin, xanthene, cyanin, pyrene, phthalocyanine, Alexa, Squaring dye, a combination producing energy transfer dyes and derivatives thereof; R1, R2, R3 R4 and R6 are various substituent groups, R5 is a substituent group except -C2H5, and R1, R2, R3, R4, R5 and R6 are not H simultaneously; and n is an integer between 0 and 10. Compared with the prior art, a kind of new azo linkage unit based reversible terminals is synthesized according to the invention; the kind of reversible terminals can realize high-efficiency shearing under moderate conditions, so that the kind of reversible terminals can be used for DNA sequencing; and raw materials required by synthesis are simple and easy to obtain, and reactions in the synthetic process are all conventional chemical reactions, therefore, the nucleotide can be subjected to large-scale promotion and application.

Description

Technical field [0001] The invention belongs to the technical field of DNA sequencing, and specifically relates to a fluorescent labeled nucleotide based on an azo link unit and its use. Background technique [0002] DNA sequencing technology is one of the important methods in modern biological research. After the completion of the Human Genome Project, DNA sequencing technology has developed rapidly. DNA sequencing refers to the analysis of the base sequence of specific DNA fragments, that is, the arrangement of adenine (A), thymine (T), cytosine (C) and guanine (G). The development of accurate, high-throughput, and low-cost DNA sequencing methods is of great significance to biology and medical science. [0003] Sequencing By Synthesis (SBS) is one of the next-generation DNA sequencing technologies. The synthetic sequencing method fixes a large number of template DNA fragments to be tested, and hybridizes and combines universal DNA primers on the fixed DNA sequencing template t...

Claims

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Application Information

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IPC IPC(8): C07H19/10C07H1/00C12Q1/68
CPCC07C247/10C07H1/00C07H19/10C12Q1/6869C12Q2535/122
Inventor 沈玉梅杨晴来谭连江李鑫辉邵志峰龚兵李小卫刘亚智张震
Owner SHANGHAI JIAO TONG UNIV
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