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Novel polymerase substrate, fluorescence generable polyphosphoric acid end-labeled nucleotide, and its application

A fluorescent labeling and nucleotide technology, which is applied in the direction of sugar derivatives, luminescent materials, sugar derivatives, etc., can solve the problems of difficult synthesis of long phosphate chains and indirect synthesis methods, so as to improve product yield and avoid by-products , the effect of polyphosphate chain length

Active Publication Date: 2015-08-19
CYGNUS BIOSCI BEIJING CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the synthesis of phosphate chains, Shiv Kumar et al. have made useful explorations (Nucleosides, Nucleotides & Nucleic Acids, 2005, 24, 401;), but there are still synthetic methods that are not direct and difficult to synthesize long phosphate chains (for example, more than five phosphates)

Method used

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  • Novel polymerase substrate, fluorescence generable polyphosphoric acid end-labeled nucleotide, and its application
  • Novel polymerase substrate, fluorescence generable polyphosphoric acid end-labeled nucleotide, and its application
  • Novel polymerase substrate, fluorescence generable polyphosphoric acid end-labeled nucleotide, and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Provided is a fluorescein analogue-carbon bridge-substituted fluorescein or anthracene-based fluorescein, which has the characteristics of long wavelength, fluorescence generation, simple and easy derivatization, high extinction coefficient and quantum yield, and has the advantages of Following structural formula (1):

[0041]

[0042] A fluorescent dye based on anthracene structure is characterized in that: it has a structure as shown in general formula (1), wherein R 0 may be selected from -H, phosphate, substituted phosphate; R 1 , R 5 Can be independently selected from -H, fluorine, chlorine, aryl, substituted aryl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy; R 2-4 , R 6-8 , R 11-13 Can be independently selected from -H, fluorine, chlorine, bromine, aryl, substituted aryl, heteroaryl, -CO 2 H, -CO 2 R, -SO 3 H, -SO 3 R, -CH 2 CO 2 H, -CH 2 CO 2 R, -CH 2 SO 3 H, -CH 2 SO 3 R, -CH 2 NH 2 、-CH 2 NHR, -NO 2 , C1-C...

Embodiment 2

[0052] According to the compound described in embodiment 1, it can specifically be:

[0053]

Embodiment 3

[0055] According to the compound described in embodiment 2, its synthetic method can specifically be as follows:

[0056]

[0057] Reagents and reaction conditions: i) THF, -40°C; ii) DCM, PCC, diatomaceous earth; iii) ZnMe 2 , TiCl 4 , DCM-40°C; iv) t-Buli, THF; o-tolualdehyde; v) DCM, PCC; vi) BBr 3 ;MeSO 3 h

[0058] 1). Synthesis of (2-bromo-5-methoxyphenyl)-3-methoxybenzyl alcohol 1a

[0059]

[0060] In a 250ml dry round bottom bottle equipped with a constant pressure dropping funnel, cool the 80ml dry tetrahydrofuran solution of m-methoxyphenylmagnesium chloride to -40°C, and dissolve 3-methoxy-o-bromobenzaldehyde under the protection of argon. (10.8g) of 20ml dry tetrahydrofuran solution was added dropwise into the reaction flask through a constant pressure dropping funnel, kept at -40°C and stirred for 2-6 hours, and the reaction was monitored by TLC. After the disappearance of the raw material 3-methoxy-o-bromobenzaldehyde The reaction was stopped, and 20 ...

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Abstract

The invention relates to a 5'-phosphoric acid and fluorescence end-labeled nucleotide and provides a molecular structure and a synthetic method thereof. The 5'-phosphoric acid and fluorescence end-labeled nucleotide is better recognizable to DNA (deoxyribonucleic acid) polymerases; after recognized by the polymerases and correctly combined with the same, by the aid of alkaline phosphatase, the 5'-phosphoric acid and fluorescence end-labeled nucleotide allows a fluorescent signal to quickly change from closure to opening; thus the 5'-phosphoric acid and fluorescence end-labeled nucleotide can serve as the polymerase substrate applicable to the fields such as nucleic acid sequencing.

Description

technical field [0001] The invention relates to a kind of 5'-terminal phosphoric acid-labeled nucleotide and its application in the nucleic acid base sequence determination method. Background technique [0002] In the application of DNA replication and amplification involving DNA polymerase, nucleotide sequence synthesis and determination, etc., the labeling technology of nucleotide molecules is involved. In recent years, fluorescently labeled nucleotides have been fully used in high-throughput DNA. In sequencing technology, among them, one of the labeling technologies is to connect different fluorescent groups to specific positions of nucleotide bases through long chains that can be cut, and remove the fluorescent groups through additional chemical means after the DNA completes recognition and records fluorescent signals. Mark the group, and then prepare for the next round of nucleotide introduction. This technology has been developed and matured by commercial institutions...

Claims

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Application Information

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IPC IPC(8): C07H19/207C07H19/10C07H1/00C09K11/06C12Q1/68
CPCC07H1/00C07H19/10C07H19/207C09K11/06C12Q1/6874
Inventor 段海峰黄岩谊陈子天谢晓亮
Owner CYGNUS BIOSCI BEIJING CO LTD
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