A kind of ortho-phthalaldehyde derivative phosphoramidite monomer, its synthesis method and the method for dna rapid coupling protein

A technology of phosphoramidite and o-phthalaldehyde, applied in the field of o-phthalaldehyde derivative phosphoramidite monomer and its synthesis, can solve the problems of long time required, low coupling efficiency, cumbersome operation and processing, etc. To achieve the effect of cheap synthetic raw materials and simple and feasible steps

Active Publication Date: 2019-03-12
XIAMEN UNIV
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  • Description
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Problems solved by technology

However, the above-mentioned covalent coupling method takes a long time, or the operation is cumbersome, and most of the coupling efficiency is low

Method used

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  • A kind of ortho-phthalaldehyde derivative phosphoramidite monomer, its synthesis method and the method for dna rapid coupling protein
  • A kind of ortho-phthalaldehyde derivative phosphoramidite monomer, its synthesis method and the method for dna rapid coupling protein
  • A kind of ortho-phthalaldehyde derivative phosphoramidite monomer, its synthesis method and the method for dna rapid coupling protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1 The synthesis of o-phthalaldehyde derivative phosphoramidite monomer, the steps are as follows:

[0040] Step 1: Synthesis of intermediate product 2 (OPA-OH), the synthesis route is shown in the figure below:

[0041]

[0042] In a 25 mL round bottom flask, 1 (252 mg, 1 mmol), 6-amino-1-hexanol (234 mg, 2 mmol), DCC (247.2 mg, 1.2 mmol), HOBT (162 mg, 1.2 mmol), 10 mL of solvent were sequentially added Tetrahydrofuran, under the protection of nitrogen, stirred at room temperature for 24h. After the reaction, the solvent was removed by rotary evaporation, separated and purified by silica gel column, characterized by NMR and mass spectrometry. 1 H NMR (500MHz, Methanol-D 4 ):δ=7.34-7.28(m,3H),6.29(s,1H),6.03(s,1H),3.56(t,2H),3.44-3.39(m,6H),3.15(m,2H), 3.01(t,2H),2.52(t,2H),1.53(m,2H),1.45(m,2H),1.35(m,2H),1.29(m,2H)ppm. 13 C NMR (500MHz, CDCl 3): δ=174.82,144.31,140.10,137.77,131.20,124.01,123.88,107.85,106.77,62.87,54.72,54.63,40.42,38.81,33.46,32.7...

Embodiment 2

[0046] Example 2 Synthesis and purification of nucleic acid molecules modified by o-phthalaldehyde derivatives.

[0047] Using 6-FAM-labeled CPG as a solid phase carrier, using normal DNA monomer bases as raw materials, synthesize DNA sequences from the 3' end to the 5' end on a DNA synthesizer, and finally modify o-phthalaldehyde at the 5' end Derivative phosphoramidite monomer. The specific synthetic sequence is as follows: 5'-X(T) 10 Y-3', wherein X is the product 3, and Y is 6-FAM. After DNA synthesis, transfer the above CPG to a 1.5mL Eppendorf tube, add 0.4mL methylamine / ammonia water (v / v=1:1), incubate at 65°C for 30min, and cut the DNA from the CPG. Then remove the methylamine / ammonia in a vacuum, dissolve it with 0.5mL 0.1mol / L triethylamine acetate (TEAA), and use C18 reverse-phase high-performance liquid chromatography to purify, as Figure 4 . The obtained product is vacuum-dried, dissolved in ultra-pure water, desalted and purified with a gel filtration colum...

Embodiment 3

[0048] Example 3 DNA was purified by reverse-phase high-performance liquid chromatography.

[0049] The experiment adopts the gradient elution procedure, using 0.1mol / L TEAA (A phase) and 95% acetonitrile (B phase) as the mobile phase, and using C18 reverse phase chromatographic column to purify DNA. The gradient elution procedure is as follows: 0-3 min, 100% A; 3-35 min, A gradually decreased from 100% to 50%. Since the 3' end of the product DNA was labeled with fluorescein, the product was monitored simultaneously with 260nm and 490nm absorption peaks. From Figure 4 As a result, it can be seen that the product was separated when the retention time was 13 min.

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Abstract

The invention relates to an o-phthaldehyde derivative phosphoramidite monomer, a synthesizing method and a method for quick coupling of DNA (deoxyribonucleic acid) and protein, and relates to quick coupling of DNA and protein. A molecular formula of the o-phthaldehyde derivative phosphoramidite monomer is C28H46N3O6P. The synthesizing method comprises the following steps of firstly, enabling o-phthaldehyde carboxylic acid derivative (OPA-COOH) and 6-amino-1-hexanol to react, so as to prepare the o-phthaldehyde hydroxyl derivative (OPA-OH); then, under the conditions of nitrogen protection, a dichloromethane solvent and N,N-diisopropylethylamine, reacting with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite for 2h at room temperature; separating by silica gel column chromatography, so as to obtain the o-phthaldehyde derivative phosphoramidite monomer. The reaction characteristic of adjacent aldehyde groups of the o-phthaldehyde derivative and the amino groups on the protein lysine residue groups quickly and efficiently generating phthalimidine is utilized, so that the purpose of quick coupling of OPA-DNA and natural protein is realized. Compared with other coupling methods, the method has the advantages that the selectivity is realized, the operation is quick, and the efficiency is high.

Description

technical field [0001] The invention relates to a phthalaldehyde derivative phosphoramidite monomer and a synthesis method thereof, in particular to a method for fast coupling of DNA and protein. Background technique [0002] The modification of natural proteins is of great significance to the field of bioanalysis research. For example, in biosensors, the coupling of DNA and proteins is usually required. Traditional DNA-protein coupling methods are mainly divided into two categories, namely non-covalent coupling and covalent coupling. The most common non-covalent coupling method is through the non-covalent binding of biotin and streptavidin. Due to their very strong non-covalent binding ability, they are widely used in the field of biological analysis, such as enzyme-linked immunosorbent adsorption. Assay (ELISA). However, the above-mentioned coupling system is cumbersome, requires the help of streptavidin with a relatively large molecular weight, and the modification ste...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07F9/655C07K1/113
CPCC07F9/65517C07K1/113
Inventor 杨朝勇马艳丽黄迪刘文丽毛瑜朱志
Owner XIAMEN UNIV
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