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Design ad synthesis method for DTPA (Diethylene Triamine Pentacetic Acid) analogue for paramagnetic labeling of proteins

A paramagnetic labeling and protein technology, which is applied in the analysis of electron paramagnetic resonance, the preparation method of peptides, organic chemistry, etc. And other issues

Inactive Publication Date: 2014-07-30
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this technique itself has certain limitations: (1) The sample must be crystal, but not all samples are easy to crystallize, and the crystal state is not easy to obtain under physiological conditions (pH, salt concentration, temperature, etc.); (2) The structure of the protein sample in the solution is actually a process of constant change, and once crystallized, it is only one of the forms of the protein, which cannot truly reflect the specific form of the protein in the solution; (3) Although X-ray The temperature factor measured by the crystal diffractometer can reflect the flexibility of biological macromolecules, but cannot reflect the speed of its dynamic changes
The dihedral angle provides the relative orientation between atoms, which can only provide short-range structural information, and it is not easy to obtain the relative orientation between protein internal domains
[0016] (3) Single optical purity
After the label is coordinated with the lanthanide metal ion, if multiple isomers are produced, multiple sets of signal peaks will appear in the NMR spectrum, which will bring difficulties to the analysis of the spectrum

Method used

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  • Design ad synthesis method for DTPA (Diethylene Triamine Pentacetic Acid) analogue for paramagnetic labeling of proteins
  • Design ad synthesis method for DTPA (Diethylene Triamine Pentacetic Acid) analogue for paramagnetic labeling of proteins
  • Design ad synthesis method for DTPA (Diethylene Triamine Pentacetic Acid) analogue for paramagnetic labeling of proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Synthetic label

[0047] (1) S-trityl-L-cysteine, namely figure 2 Synthesis of Compound 7 in

[0048] Add 10.0g (63.50mmol) of L-cysteine ​​hydrochloride, 15.80g (56.68mmol) of triphenylchloromethane, and 75.0ml of DMF into a 250ml single-necked bottle, stir at room temperature for 20-30h, pour it into 10 % NaOAc solution, a white solid precipitated, filtered, washed the filter cake with deionized water, recrystallized the filter cake in anhydrous acetone, filtered after cooling, washed the filter cake with anhydrous acetone and anhydrous ether, and dried by infrared , to obtain white powder 7.21g, yield: 34.8%. 1 H-NMR (400 MHz, DMSO-d 6 ) δ ppm: 7.24-7.37 (15H, m), 2.92 (1H, dd, J = 9.2Hz, 4.2Hz), 2.58 (1H,dd, J = 12.5Hz, 4.2Hz), 2.40 (1H,dd, J = 12.5Hz, 9.2Hz).

[0049] (2) S-trityl-L-cysteine ​​tert-butyl ester, namely figure 2 Synthesis of Compound 8 in

[0050] Add 1.51g (4.16mmol) of 7 and 24.5ml of tert-butyl acetate into a 100ml th...

Embodiment 2

[0059] Example 2 Paramagnetic label DTPA-CH 2 Application of S-SPy

[0060] The protein used to attach the tag is 15 The mutant of N-labeled human ubiquitin protein ubiquitin (Ubi): ubiquitin G47C, which uses the cysteine ​​sulfhydryl group of the mutant to exchange the disulfide bond of the paramagnetic label to achieve the purpose of connection. image 3 is the connection mode of protein and paramagnetic label. The specific connection process is as follows:

[0061] (1): Configure 50 mM DTPA-CH 2 S-SPy label solution: Weigh it quantitatively, add MQ ultrapure water, and dissolve.

[0062] (2): Gradually add the protein dropwise to five times the amount of the labeling solution to ensure that the reaction pH is about 6.4, react for 1~2 hours, pass through the PD10 desalting column, remove small molecules, and obtain the final product, which is the protein solution with the label attached. If the label cannot be removed, it can be separated by DEAE anion exchange column. ...

Embodiment 3

[0065] Example 3 Paramagnetic label DTPA-CH 2 Application of S-SPy

[0066] The reaction pH was 3.0, and the rest of the steps were the same as in Example 2.

[0067] The result of embodiment 2 and embodiment 3 is as Figure 4 , 5 , 6 and 7 show:

[0068] Figure 4 0.1 mM ubiquitin G47C and 0.1 mM complex ubiquitin G47C-DTPA at 25℃, pH=6.4 1 H- 15 Overlay of N HSQC spectra, gray indicates the spectrum of the complex ubiquitin G47C-DTPA. The amino acid residues marked in the spectrum are residues with large chemical shifts after the protein is attached to the label, which shows that most of the amino acid residues overlap well, and only the amino acid residues near G47C have a large change, indicating that paramagnetic The introduction of tags did not have much impact on the protein structure.

[0069] Figure 5 0.1 mM ubiquitin G47C and 0.1 mM complex ubiquitin G47C-DTPA at 25℃, pH=3.0 1 H- 15 Overlay of N HSQC spectra, gray indicates the spectrum of the complex ubi...

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Abstract

The invention belongs to the field of the paramagnetic labeling of the proteins, and provides a DTPA (Diethylene Triamine Pentacetic Acid) analogue for paramagnetic labeling of proteins. The DTPA analogue with eight coordination sites is a mercaptopyridine activated paramagnetic label and can be directly used for labeling the connection of protein and protein; if lanthanide series metal ions are added dropwise to the DTPA analogue, the DTPA analogue is capable of generating huge pseudocontact shifts and residual dipolar couplings and thus can be still used even at a low pH.

Description

technical field [0001] The invention belongs to the field of protein paramagnetic labeling, and relates to the synthesis and application of DTPA analogues for protein paramagnetic labeling. Background technique [0002] The molecular structure of protein is the basis of its biological function. High-field liquid phase nuclear magnetic resonance (NMR), as one of the two main means to analyze protein structure with atomic resolution, has developed rapidly in the past two decades. The other main method is X-ray crystal diffraction. So far, X-ray crystal diffraction technology is the most important technical means to intuitively explain the spatial structure information of biological macromolecules. It has made outstanding contributions to the development of molecular biology. contribute. However, this technique itself has certain limitations: (1) The sample must be crystal, but not all samples are easy to crystallize, and the crystal state is not easy to obtain under physiolo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D213/71C07K1/13G01N24/10
CPCC07D213/71C07K1/13G01N24/10
Inventor 苏循成王金涛裴莹莹
Owner NANKAI UNIV
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