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 b

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

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Example Embodiment

[0045] Example 1:

[0046] a) Synthesis of Di tert butyl n - (2-bromoethyl) iminodiacetic acid, i.e. synthesis of compound 3;

[0047] Dissolve 2-bromoethylaminohydrobromate (20.5g, 100mmol) into 100mldmf, add 100ml2 tert butyl bromoacetate (30g, 150mmol) DMF solution under stirring at room temperature, and then add dipea (70ml, 400mmol). The reaction was stirred at room temperature for 9 ~ 10 hours, filtered, concentrated and passed through the column. The target product compound 3 was 23g colorless oil, and the yield was about 67%. 1 HNMR(400MHz,CDCl 3 ) δ ppm: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-triphenylmethyl-d-cysteine tert butyl ester, i.e. synthesis of compound 5;

[0049]S-triphenylmethyl-d-cysteine (6.0g, 16.5mmol) was added to 100ml tert butyl acetate, perchloric acid (4.8ml, 70%) was added dropwise under ice bath, and the reaction was stirred. After dropping, stir the reaction at room temperature for 1 ~ 2 hours, and st...

Example Embodiment

[0056] Example 2. Application of paramagnetic probe py-d-cys-dtpa

[0057]1) Quantitatively weigh the paramagnetic probe (i.e. compound 8) prepared in example 1, dissolve it in water and prepare 30mm py-d-cys-dtpa aqueous solution. The water used is MQ ultrapure water.

[0058] 2) 0.5ml 1.0mm ubiquitin e18c, ubiquitin a28c, ubiquitin g47c and ubiquitin e64c protein solutions were prepared respectively, and the buffer solution was 20mm Tris Cl (pH = 7.5).

[0059] 3) Add 3 times of equivalent label to the prepared protein solution at room temperature and react for 3 hours. The protein reaction is complete by mass spectrometry（ Figure 2 ）The protein was purified by PD10 column to obtain the site modified protein（ Figure 1 )。

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

[0061] All nuclear magnetic experiments were collected on the QCI ultra-low temperature probe of bruker-600mhz nuclear magnetic instr...

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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 ...

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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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