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Molecular glue-based fluorescently labeled nucleotides and their use in dna sequencing

A fluorescent labeling, nucleotide technology, applied in the fields of chemical synthesis and biochemistry, which can solve problems such as poor selectivity

Active Publication Date: 2017-12-08
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when this method is used to connect small molecule compounds, a series of difficulties are encountered. First, the selectivity of the reaction is very poor. Through repeated screening and optimization of reaction conditions, the method of connecting specific small molecule compounds under specific conditions is finally obtained. , can still get good selectivity cross-EU product

Method used

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  • Molecular glue-based fluorescently labeled nucleotides and their use in dna sequencing
  • Molecular glue-based fluorescently labeled nucleotides and their use in dna sequencing
  • Molecular glue-based fluorescently labeled nucleotides and their use in dna sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] This embodiment relates to a fluorescently labeled nucleotide based on molecular glue, that is, a reversible terminal whose structural formula is the following formula (II):

[0085]

[0086] The corresponding synthetic route is as Image 6 Shown; specifically include the following steps:

[0087] 1 compound dUTP (AP 3 )-A synthesis

[0088] 1.1dUTP (AP 3 )Synthesis:

[0089]

[0090] 1.1.1 Compound trifluoroethylpropynylamine F 2 Synthesis

[0091] 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 under an ice-water bath, add propargylamine (60mmol, 3.3042g), stir for 15 minutes and then slowly add methyl trifluoroacetate (86.7mmol, 11.0957g) for 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,...

Embodiment 2

[0129] This embodiment relates to a fluorescently labeled nucleotide based on molecular glue, that is, a reversible terminal whose structural formula is the following formula (III):

[0130]

[0131] Specifically include the following steps:

[0132] 1 Synthesis of compound dUTP-A-click

[0133] 1.1 Compounds A-N 3 Synthesis

[0134] Compound A-N 3 The synthetic route of figure 2 Shown: Under basic conditions, compound A is subjected to amidation reaction with 2-azidoethylamine whose terminal is an amino group to obtain compound A-N 3 ;

[0135] The steps are as follows: Weigh compound A (0.841g, 0.1mmol) and dissolve 5ml DMF in a 10ml single-necked flask, add NMM (N-methylmorpholine) (200μL, 2mmol), HATU (2- (7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate) (0.57g, 1.5mmol) was activated for 30min, and 2-azidoethylamine ( 129mg, 1.5mmol) stirred for 1h, warmed up to room temperature and reacted for 8h, stopped the reaction, added an appropriate...

Embodiment 3

[0153] This embodiment relates to a fluorescently labeled nucleotide based on molecular glue, that is, the structural formula is a reversible terminal shown in the following formula (IV):

[0154]

[0155] Its synthetic concrete steps are as follows:

[0156] 1 compound dUTP (AP 3 )-A synthesis

[0157] The synthesis of dUTP(AP3)-A refers to Example 1.

[0158] 2 Synthesis of compound TAMRA-B-click

[0159] 2.1 Compounds B-N 3 Synthesis

[0160] B-N 3 The synthetic route of is as follows:

[0161]

[0162] The steps are as follows: Weigh bromoethanediazide (0.3g, 2mmol), B (1.14g, 1.5mmol) in a dry single-necked flask of 10ml, add NMM (N-methylmethanol) under ice-water bath and nitrogen protection phenoline) (200μL, 2mmol), dry DMF10ml, reflux at 120°C for 18h, stop the reaction, extract the reaction solution with dichloromethane, rotary evaporate to obtain 1.3g of crude product, column chromatography 1.0mg, and obtain B-N 3 , yield 82%. 1 H NMR (500MHz, CDCl 3...

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Abstract

The invention discloses a fluorescence labelled nucleotide based on a molecular glue and a use thereof in DNA sequencing. The structure formula of the fluorescence labelled nucleotide is shown in a formula (I) in the specification, wherein R1 is shown in the specification, R2 is fluorescein or shown in the specification, and dNTP is ribonucleoside triphosphote which contains four different base groups; the fluorescein is selected from one of the BODIPY, rhodamine, coumarin, xanthene, cyanin, pyrene, phthalocyanine, alexa, a squarene dye, a composition for generating energy transfer dye and the derivatives thereof. The fluorescence labelled nucleotide can be used for DNA sequencing; simultaneously the raw materials for synthesizing the fluorescence labelled nucleotide are simple and easy to obtain and the fluorescence labelled nucleotide can be used for large-scale popularization. The biological assessment result shows that all the requirements of the high-throughput sequencing biochemical reaction can be satisfied by the reversible terminal, and the reversible terminal has good practical prospect.

Description

technical field [0001] The invention relates to the fields of chemical synthesis and biochemistry, in particular to a class of fluorescently labeled nucleotides based on molecular glue and its application in DNA sequencing. Background technique [0002] DNA sequencing technology is one of the important means of modern life science and medical research. DNA sequencing started with Sanger sequencing technology (generation sequencing) in 1977, and has developed rapidly in the past thirty years. The throughput of sequencing has been greatly increased and the cost has dropped sharply. Some people even think that the speed of its development has broken the existing Moore's Law budget in the semiconductor industry. The emergence of the second-generation high-throughput parallel sequencing technology is a concentrated expression of the rapid development of sequencing technology. Using first-generation sequencing technology, the Human Genome Project (HGP) spent $3 billion to sequen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07H19/10C07H1/00C09K11/06C12Q1/68
CPCC07H1/00C07H19/10C09K11/06C09K2211/1022C09K2211/1081C12Q1/6869C12Q2535/122
Inventor 沈玉梅龚兵谭连江邵志峰杨晴来李小卫姜玉张震
Owner SHANGHAI JIAO TONG UNIV
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