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Synthesis method and application of chiral paramagnetic probe Py-D-Cys-DTPA

A technology of py-d-cys-dtpa and synthesis method is applied in the field of synthesis of chiral paramagnetic probe Py-D-Cys-DTPA, which can solve the problem that the rigidity of paramagnetic probe is affected by protein modification sites and the like , to achieve the effect of strong rigidity and great universality

Pending Publication Date: 2022-05-13
NANKAI UNIV
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Problems solved by technology

[0008] The purpose of the present invention is to solve the problem that the rigidity of the paramagnetic probe is greatly affected by the protein modification site, and to provide a synthesis method and application of a novel chiral paramagnetic probe Py-D-Cys-DTPA

Method used

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  • Synthesis method and application of chiral paramagnetic probe Py-D-Cys-DTPA
  • Synthesis method and application of chiral paramagnetic probe Py-D-Cys-DTPA
  • Synthesis method and application of chiral paramagnetic probe Py-D-Cys-DTPA

Examples

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Effect test

Embodiment 1

[0046] a) Synthesis of N-(2-bromoethyl) di-tert-butyl iminodiacetate, i.e. the synthesis of compound 3;

[0047] 2-Bromoethylamine hydrobromide (20.5g, 100mmol) was dissolved in 100mL of DMF, and 100mL of tert-butyl 2-bromoacetate (30g, 150mmol) in DMF was added with stirring at room temperature, followed by DIPEA (70mL, 400mmol). The reaction was stirred at room temperature for 9-10 hours, filtered, concentrated, and passed through a column to obtain 23 g of the target compound 3 as a colorless oil with a yield of about 67%. 1 HNMR (400MHz, CDCl 3 )δppm: 3.46(s, 4H), 3.42(t, J=7.6, 2H), 3.12(t, J=7.6Hz, 2H), 1.45(s, 18H).

[0048] b) Synthesis of S-trityl-D-cysteine ​​tert-butyl ester, i.e. the synthesis of compound 5;

[0049] S-trityl-D-cysteine ​​(6.0 g, 16.5 mmol) was added to 100 mL of tert-butyl acetate, perchloric acid (4.8 mL, 70%) was added dropwise under ice cooling, and the reaction was stirred. After dropping, stir and react at room temperature for 1 to 2 hours...

Embodiment 2

[0056] Embodiment 2, application of paramagnetic probe Py-D-Cys-DTPA

[0057] 1) Quantitatively weigh the paramagnetic probe prepared in Example 1 (ie, compound 8), dissolve it in water, and prepare a 30 mM Py-D-Cys-DTPA aqueous solution, and the water used is MQ ultrapure water.

[0058] 2) 0.5 mL of 1.0 mM ubiquitin E18C, ubiquitin A28C, ubiquitin G47C and ubiquitin E64C four protein solutions were prepared respectively, and the buffer solution was selected as 20 mM Tris-Cl (pH=7.5).

[0059] 3) Add 3 times the equivalent of the label to the prepared protein solution at room temperature, react for 3 hours, and mass spectrometry detects that the protein reaction is complete ( figure 2 ), and then purified with a PD10 column to obtain a site-specific modified protein ( figure 1 ).

[0060] 4) Through the titration of rare earth ions, collect 1 H- 15 N HSQC spectrum ( image 3 ), the fitted tensor ( Figure 4 ).

[0061] All NMR experiments were collected on the Bruker-...

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Abstract

The invention provides a synthesis method and application of a chiral paramagnetic probe Py-D-Cys-DTPA (Diethylene Triamine Pentacetate Acid). The synthesis method comprises the following steps: esterifying S-trityl-D-cysteine in a tert-butyl acetate solution, and introducing a side chain through nucleophilic substitution reaction in acetonitrile under an alkaline condition. And hydrolyzing the intermediate product by trifluoroacetic acid, and finally activating the hydrolysis product in methanol by using 2-mercaptopyridine to synthesize. The probe shows excellent properties in a paramagnetic labeling technology: 1) the probe has relatively high reaction activity with protein, and has relatively high selectivity to sulfydryl; 2) the probe is stable and good in water solubility; 3) the compound contains 8 coordination sites and can be stably combined with rare earth ions; and 4) the method has strong rigidity, and is less affected by the microenvironment of the protein connection site.

Description

technical field [0001] The invention belongs to the technical field of protein paramagnetic labeling, in particular to a synthesis method and application of a chiral paramagnetic probe Py-D-Cys-DTPA. Background technique [0002] Protein paramagnetic labeling technology is one of the important means to study the structure, dynamic changes and functions of proteins. It can obtain long-range protein structure information at the atomic resolution level under near-physiological conditions. About 40% of natural proteins bind metal ions or use metal ions as cofactors (Chinese Science: Chemistry, 2019, 49:1–15), but most of these metal ions do not have paramagnetic properties. Therefore, before using paramagnetic technology to study proteins, it is first necessary to construct a paramagnetic center and introduce a paramagnetic substance. [0003] In nature, rare earth ions, transition metal ions and nitroxide radicals are commonly used paramagnetic substances and are widely used ...

Claims

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Application Information

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IPC IPC(8): C07D213/71C07K1/13
CPCC07D213/71C07K1/13C07B2200/07
Inventor 苏循成李斌
Owner NANKAI UNIV
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