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Reversible terminal and synthesis and use in DNA synthesis sequencing thereof

A terminal and compound technology, applied in the fields of chemical synthesis and biochemistry, can solve problems that cannot be practically applied

Active Publication Date: 2013-05-08
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The linking units that have been reported so far include photocleavage, Pd catalytic cracking, fluoride cleavage, etc., but these linking units are currently limited to basic research and cannot be practically applied (Accounts of Chemical Research 2010, 43, 551-563.)

Method used

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  • Reversible terminal and synthesis and use in DNA synthesis sequencing thereof
  • Reversible terminal and synthesis and use in DNA synthesis sequencing thereof
  • Reversible terminal and synthesis and use in DNA synthesis sequencing thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] The structural formula of the reversible terminal in this embodiment is shown in the following formula (II):

[0078]

[0079] The corresponding synthetic route is as figure 2 Shown; specifically include the following steps:

[0080] 1.1 Compound F 2 Synthesis

[0081] Methyl trifluoroacetate reacts with propargylamine in an organic solvent to obtain compound F 2 , specifically: add 60ml of methanol to a single-necked bottle, stir in an ice-water bath, add propargylamine (60mmol, 3.3042g), stir for 15 minutes, then slowly add methyl trifluoroacetate (86.7mmol, 11.0957g), 10 minutes Afterwards, the ice-water bath was removed, and the reaction was carried out at room temperature for 24 hours. The reaction was monitored with a TLC plate, PE:EA=8:1, baking plate, Rf=0.5 to produce a new spot as product F2. Distillation under reduced pressure (51°C, 280Pa) yielded 3.53g with a yield of 39%.

[0082] 1 H NMR (CDCl 3 , 300MHz): δ2.32(t, J=4.0Hz, 1H), 4.13-4.15(m, 2H...

Embodiment 2

[0108] The structural formula of the reversible terminal in this embodiment is shown in the following formula (II):

[0109]

[0110] The corresponding synthetic route is as Figure 5 Shown; specifically include the following steps:

[0111] 2.1 Compound F 2 , F 3 The synthesis is the same as in Example 1

[0112] 2.2 Synthesis of compound G1

[0113] Take 23mg F 3 (0.06mmol) in a 10mL single-necked bottle, add 1mL of methanol to dissolve, add 0.1mL of concentrated ammonia (6mmol), and stir overnight at room temperature. TLC plate monitoring: DCM:MeOH=3:1, product G1 Rf=0.15. Separation adopts TLC plate chromatography, MeOH:EA:NH3=6:6:1, and collects Rf=0.6 ultraviolet color region. ESI-HRMS: cals for C 12 h 15 N 3 o 5 [M] 281.1012, found 281.1015.

[0114] In the above synthesis, the ammonia water added can be any value in 3-6 mmol.

[0115] 2.3 Synthesis of Compound G2

[0116] Take 8.5mg G1 (0.03mmol) and dissolve it with 0.5mL methanol; take 9.4mg SPDP (0....

Embodiment 3

[0128] The structural formula of the reversible terminal of this embodiment is shown in formula (III):

[0129]

[0130] The corresponding synthetic route is as Figure 7 As shown, the specific steps are as follows:

[0131] 3.1 Synthesis of Compound N-1

[0132] Take a 100ml one-mouth bottle, add 0.75g (6.6mmol) of cysteamine hydrochloride, dissolve it with 4ml of methanol, and add 2.04g (6.6mmol, 50% aqueous solution, Dissolve in 3ml of methanol) and 1.85ml of TEA (13.2mmol) mixture, remove the ice-water bath after 30min, and stir at room temperature. The reaction progress was tracked by TLC, and the reaction was stopped after 24 h. The solvent was spun out, and plate chromatography, MeOH:EA=1:1, gave 44 mg of the product as a yellow oily liquid.

[0133] 1 H NMR (D 2 O, 400MHz): δ2.92(t, J=6.0Hz, 2H), 3.00(t, J=6.4Hz, 2H), 3.40(t, J=6.4Hz, 2H), 3.87(t, J=6.0 Hz, 2H).

[0134] In the above synthesis, the added 2-hydroxyethyl disulfide can be any value in 6.6-13.2 m...

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Abstract

The invention discloses a reversible terminal and synthesis and use in DNA synthesis sequencing thereof. The structural formula of the reversible terminal is described according to formula (I), wherein R1 is fluorescein and R2 is a connection unit. The cracking reversible terminal of the invention is available for DNA synthesis sequencing. At the same time, as synthesis required material is easily available and synthesis processes are all conventional chemical reactions, the reversible terminal disclosed by the invention is applicable for large-scale promotion and has good practical prospect due to a biological evaluation result showing that the reversible terminal is capable of totally satisfying biochemical reaction requirements of high-energy sequencing.

Description

technical field [0001] The invention relates to the fields of chemical synthesis and biochemistry, in particular to a type of reversible terminal and its synthesis and application in DNA synthesis sequencing. Background technique [0002] DNA sequencing technology is one of the important means in modern biological research. After the completion of the Human Genome Project, DNA sequencing technology has developed rapidly. DNA sequencing (DNA sequencing) refers to the analysis of the base sequence of a specific DNA fragment, 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 biological and medical sciences. [0003] Sequencing By Synthesis (SBS) is one of the next-generation DNA sequencing technologies. The sequencing by synthesis method immobilizes a large number of template DNA fragments to be tested, hybridizes and combines univer...

Claims

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Application Information

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IPC IPC(8): C07H19/10C07H1/00C12Q1/68
Inventor 沈玉梅赵小东邵志峰龚兵汤道年李小卫彭丽娜邢宇洋庄园黎庆伍新燕
Owner SHANGHAI JIAO TONG UNIV
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