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Thioketal linking unit and its synthesis method and application

A thioketal and linking unit technology, applied in the field of thioketal linking unit and its synthesis, can solve the problems affecting the extension efficiency of reversible terminators, DNA strand damage, large molecular traces, etc. Deficiencies and weaknesses, the effect of avoiding the use of acidic conditions

Active Publication Date: 2019-01-11
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] When a reversible terminator based on an acid-sensitive linking unit is used for DNA sequencing, it will inevitably cause damage to the DNA chain to a certain extent, and the reversible terminator based on the azo linking unit will leave a large molecular trace on the DNA chain after breaking. To a certain extent, it affects the extension efficiency of the next reversible terminator; and the linking unit based on the thioketal structure can overcome the above shortcomings to a certain extent, so it is expected to bring new opportunities in the field of DNA sequencing

Method used

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  • Thioketal linking unit and its synthesis method and application
  • Thioketal linking unit and its synthesis method and application
  • Thioketal linking unit and its synthesis method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Embodiment 1, when R 1 = R 2 =Me, R=-COOH, the synthesis of this type of linking unit

[0066] The schematic diagram of the synthesis of the oxidation-sensitive thioketal linking unit of this example is as follows figure 1 As shown, the specific steps are as follows:

[0067] The first step, the synthesis of compound 5, see the following formula:

[0068]

[0069] Under nitrogen protection, 2-mercaptoethanol hydrochloride (10g, 88mmol) and triethylamine (24mL, 0.17mol) were dissolved in methanol (0.25L), the mixture was cooled to 0°C, and then three Ethyl fluoroacetate (10mL, 84mmol) was added to the reaction mixture, reacted at 0°C for 3h and then continued to react at room temperature for 12h; the solvent was removed with a rotary evaporator, the residue was dissolved in ethyl acetate (0.10L), and water (50mL ) and saturated brine (50 mL) were washed successively, the organic phase was dried over sodium sulfate, and purified by column chromatography to obtai...

Embodiment 2

[0082] Embodiment 2, when R 1 with R 2 Together to form a cyclohexyl group, when R=-COOH, the synthesis of this type of linking unit

[0083] The first step, the synthesis of compound 5: the same as in Example 1.

[0084] The second step, the synthesis of compound 6-1:

[0085]

[0086] Under nitrogen protection, 2-mercaptotrifluoroacetamide 5 (5.7g, 33mmol), methyl 3-mercaptopropionate (4.4g, 37mmol) and cyclohexanone (34mmol) were dissolved in acetonitrile (50mL), and the reaction mixture was cooled to Add boron trifluoride diethyl ether (13mL, 0.11mol) after 0°C, continue the reaction at 0°C for 2h, add 15% sodium carbonate solution (0.30L) to the reaction mixture, extract with ethyl acetate (100mL×3), organic The phase was washed with 5% sodium carbonate solution (0.30 L), dried, and the solvent was spun off, separated and purified by silica gel column to obtain the expected product thioketal 6-1.

[0087] The third step, the synthesis of compound 10-1:

[0088]...

Embodiment 3

[0090] Embodiment 3, when R 1 with R 2 Together to form a cyclopentyl group, when R=-COOH, the synthesis of this type of linking unit

[0091] The first step, the synthesis of compound 5: the same as in Example 1.

[0092] The second step, the synthesis of compound 6-2:

[0093]

[0094] Under nitrogen protection, 2-mercaptotrifluoroacetamide 5 (5.7g, 33mmol), methyl 3-mercaptopropionate (4.4g, 37mmol) and cyclopentanone (34mmol) were dissolved in acetonitrile (50mL), and the reaction mixture was cooled to Add boron trifluoride diethyl ether (13mL, 0.11mol) after 0°C, continue the reaction at 0°C for 2h, add 15% sodium carbonate solution (0.30L) to the reaction mixture, extract with ethyl acetate (100mL×3), organic The phase was washed with 5% sodium carbonate solution (0.30 L), dried, and the solvent was spun off, separated and purified by silica gel column to obtain the expected product thioketal 6-2.

[0095] The third step, the synthesis of compound 10-2:

[009...

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Abstract

The invention discloses a thioketal connection unit, a synthetic method and a purpose thereof. The thioketal connection unit has a structure shown as a formula (I) in the specification, wherein, R=-OH or -COOH; R1 and R2 can satisfy one of the following conditions: 1) R1=R2=methyl; or, 2) R1=phenyl, p-methoxyphenyl, 2,4,6-trimethoxyphenyl, naphthyl or p-methoxynaphthyl, R2=H; or, and 3) R1, R2 co-forms cyclohexyl or cyclopentyl. The unit is connected with nucleotide and fluorescein to obtain fluorescein-labeled nucleotide for DNA sequencing. Compared with the prior art, when a reversible terminator of the connection unit is used for DNA sequencing, extension efficiency is close to 100%, when a template is continuous several same bases, one reversible terminator is extended at one time; the raw materials required for synthesis are simple and easily available, the synthesis processes are conventional chemical reactions, and the method can be used for large-scale popularization and usage.

Description

technical field [0001] The invention belongs to the fields of chemical synthesis and biochemistry, and relates to a linking unit that can be used for DNA sequencing, in particular to a thioketal linking unit and its synthesis method and application. 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 biology and medicine. [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, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07C323/52C07C323/25C07H19/10C07H19/14C07H1/00C12Q1/6869
CPCY02P20/55
Inventor 沈玉梅王雪里邵志峰龚兵谭连江
Owner SHANGHAI JIAOTONG UNIV
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