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Selenium or thiothymine nucleoside-5 '-triphosphoric acid and synthesis method thereof

A technology of thiothymidine and thymidine, which is applied in the fields of modified triphosphate synthesis and nucleic acid molecule detection, can solve the problems of accurate detection by instruments, flooding, weak signal of low-copy nucleic acid molecules, etc., and achieves pairing accuracy The effect of improving, improving accuracy and sensitivity

Pending Publication Date: 2021-06-22
纽奥维特(成都)生物科技有限公司 +1
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  • Abstract
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Problems solved by technology

[0004] The biggest pain point of the existing technology is that the detection sensitivity of pathogens, tumors, diseases and environmental nucleic acid samples is not high enough for low copy number, resulting in false negative results
This is because the presence of mismatches (especially typical T / dG and T / rG wobble mismatches) in the detection process of the prior art will cause the signal of low-copy nucleic acid molecules to be weak, and the background noise will overwhelm them. Submerged, cannot be accurately detected by instruments

Method used

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  • Selenium or thiothymine nucleoside-5 '-triphosphoric acid and synthesis method thereof
  • Selenium or thiothymine nucleoside-5 '-triphosphoric acid and synthesis method thereof
  • Selenium or thiothymine nucleoside-5 '-triphosphoric acid and synthesis method thereof

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Embodiment 1

[0068] according to Figure 1-13 As shown, this embodiment provides a kind of selenothymidine-5'-triphosphate and its synthesis method, which is characterized in that it comprises the following synthesis steps:

[0069] Step 1: Using 5′-DMTr-2-thiothymidine as compound 1, using CH 3Alkylation of the 2-thiol functional group by I to activate the 2-thiol moiety affords compound 2

[0070] Step 2: Compound 2 was reacted with freshly prepared NaSeH to complete the selenium substitution, and compound 3 was obtained in 80% yield

[0071] Step 3: Compound 3 is deprotected with trichloroacetic acid to obtain Se T, the compound 4

[0072] Step 4: Conversion of compound 4Se to Se TTP, the compound 5Se

[0073] Step 5: Purify compound 5Se and characterize it by nuclear magnetic resonance, mass spectrometry and high performance liquid chromatography to confirm its structure and purity, and then use Se TTP and DNA polymerase for DNA enzymatic synthesis, the DNA polymerase can use Kle...

Embodiment 2

[0099] Such as figure 1 As shown, this example provides a chemical synthesis method of thiothymidine-5'-triphosphate, comprising the following synthesis steps:

[0100] Step 1: using 5'-DMTr-2-thiothymidine as the initial reactant, that is, compound 1, deprotecting reaction between compound 1 and trichloroacetic acid to obtain S T, that is, compound 4S;

[0101] Step 2: Transform compound 4S into S TTP, namely compound 5S;

[0102] Step 3: Characterize compound 5S after purification to confirm its structure and purity, and then use S TTP and DNA polymerase carry out DNA enzymatic synthesis; The present invention is through the method of innovative synthesis triphosphate, for example 2-thiothymidine-5'-triphosphate ( S TTP) established a sulfur atom-specific modification strategy (SAM, Sulfur Atom-specific Modification of Nucleic Acids) to suppress T / G mismatches in DNA polymerization, improve the specificity of base pairing, and proved that the SAM method can improve polym...

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Abstract

The invention discloses selenium or thiothymine nucleoside-5 '-triphosphoric acid and a synthesis method of the selenium or thiothymine nucleoside-5'-triphosphoric acid. The synthesis method comprises the following partial synthesis steps: a deprotection reaction is carried out on a compound 3 or 1 and trichloroacetic acid to respectively obtain SeT (namely 4Se) or ST (namely 4S); then, the compound 4Se and the compound 4S are respectively converted into a compound 5Se and a compound 5S through a one-pot synthesis method, SeTTP is the compound 5Se, and STTP is the compound 5S; next, the compounds 5Se and 5S are purified, the purified compounds 5Se and 5S are characterized to confirm the structure and purity of the compounds 5Se and 5S, and then DNA enzymatic synthesis is carried out by using SeTTP or STTP and DNA polymerase to obtain a compound 6, namely Se-DNA or S-DNA or selenium sulfo Se / S-DNA; according to the invention, a selenium or sulfur atom specific modification strategy SAM is established by innovatively synthesizing SeTTP and STTP and innovatively synthesizing DNA polymerase, so that T / G mismatch in DNA polymerization is inhibited, the specificity of base pairing is improved, and the SAM method is proved to be capable of improving the accuracy and sensitivity of polymerase reaction, nucleic acid molecule recognition and molecule detection.

Description

technical field [0001] The invention relates to the technical field of modified triphosphate synthesis and nucleic acid molecule detection, in particular to a method for selenium or thiothymidine-5'-triphosphate and its nucleic acid synthesis and nucleic acid molecule detection. Background technique [0002] Nucleic acid molecular detection and nucleic acid sequencing are important foundations and basis for the detection and identification of pathogens, tumors, diseases and the environment, and epidemic control. Among them, base pairing is the basis of nucleic acid molecular recognition, and they are of great significance in the discovery and practical application of mechanisms including nucleic acid diagnosis, molecular sequence recognition, sequencing analysis, genetic information storage, DNA replication, RNA transcription and protein translation, etc. . In addition, the normal and accurate pairing and stacking of DNA bases (T / A and C / G) determine the structure and stabi...

Claims

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Application Information

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IPC IPC(8): C07H19/10C07H19/073C07H1/00C12P19/30
CPCC07H19/10C07H19/073C07H1/00C12P19/305
Inventor 黄震李洋胡若多
Owner 纽奥维特(成都)生物科技有限公司
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