Polyglycol modifying ferric iron deuterohemin short peptide compound and production method thereof

A technology of polyethylene glycol succinimidyl ester and polyethylene glycol, applied in the biological field, can solve problems such as low bioavailability, and achieve the effects of simple modification method, good stability and high yield

Inactive Publication Date: 2008-07-16
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, its bioavailability is low and it is susceptible to protease degradation

Method used

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  • Polyglycol modifying ferric iron deuterohemin short peptide compound and production method thereof
  • Polyglycol modifying ferric iron deuterohemin short peptide compound and production method thereof
  • Polyglycol modifying ferric iron deuterohemin short peptide compound and production method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Methoxy polyethylene glycol succinimide carbonate (see the general formula for the structure, k=0, the weight-average molecular weight of PEG is 20000, n≈447) (m-SC-PEG20000) to subheme hexapeptide (DhHP -6) modification:

[0034] The choice of reaction temperature: DhHP-6 solid powder is dissolved in deionized water, is mixed with the DhHP-6 deionized aqueous solution of 1mg / ml, gets 4 parts of 0.1ml DhHP-6 deionized aqueous solution (1mg / ml), with pH =7 phosphate buffered saline (PBS) was diluted to 1ml, and 20mg m-SC-PEG20000 was added respectively (the following refers to the molar ratio, and the molar ratio is approximately equal to (20 / 20000) / (0.1 / 1000)=10:1) , after completely dissolving, placed in the environment of 0°C, 4°C, 15°C, and 25°C for 1 hour, and then added glycine to terminate the reaction. Detect conversion rate with reverse phase high-performance liquid chromatography, record its conversion rate to be 37.1%, 38.5%, 47.9% and 55.1% respectively, the...

Embodiment 2

[0039] Example 2: Separation, purification and identification of modified products

[0040] Dissolve DhHP-6 solid powder in deionized water to prepare 1mg / ml DhHP-6 deionized aqueous solution, take 0.1ml DhHP-6 deionized aqueous solution (1mg / ml), and use pH=8 phosphate buffer (PBS) was diluted to 1ml, and 20mg of m-SC-PEG20000 was added. After it was completely dissolved, it was placed in an environment at 25°C. After reacting for 2 hours, glycine was added to terminate the reaction.

[0041] The upper column separation of the above reaction solution, the chromatographic conditions are as follows:

[0042] Chromatograph: BioCAD 700E;

[0043] Column specifications: prepLC, 25mmModule;

[0044] Flow rate: 10ml / min;

[0045] Mobile phase: A:H 2 O (containing 0.5% trifluoroacetic acid (TFA, v / v)), B: 90% aqueous acetonitrile (containing 0.5% trifluoroacetic acid (TFA, v / v));

[0046] Concentration gradient: elution time 0-5min, 100% A phase, elution time 5-45min, B phase gr...

Embodiment 3

[0050] Embodiment 3: the biological activity (specific activity) measurement of modification product

[0051] The microenzyme that hydrolyzes with cytochrome c, MP-9 is used as standard substance contrast, with ascorbic acid (V C ) and H 2 o 2 As the substrate, the peroxidase activity of DhHP-6 and PEG-DhHP-6 was determined, and the specific activity of DhHP-6 was 4.0×10 3 U / μmol, while the specific activity of PEG-DhHP-6 is 3.1×10 3 U / μmol (defining the amount of enzyme needed to oxidize 1 μg VC per minute as an activity unit U), the results are shown in Table 1: as can be seen from the table, PEG-modified DhHP-6 still has a high activity, indicating that this modification method is feasible.

[0052] Table 1: Specific activities of DhHP-6 and PEG-DhHP-6

[0053] sample

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Abstract

The invention belongs to the biotechnology field, which particularly relates to a deuterohemin short peptide compound modified by the Monomethoxy-Polyethylene Glycol Succinimidyl Ester derivatives and the preparation method for the compound, which adds phosphate buffer into the water solution prepared by a deuterohemin 6-mer peptide, and then reacts by adding the solid powder of active Polyethylene Glycol derivatives or the acetonitrile solution of the Polyethylene Glycol derivatives and obtains the deuterohemin 6-mer peptide modified by the Polyethylene Glycol by chromatographic separating of the reaction mixture with the reversed-phase high performance Liquid Chromatography. The deuterohemin 6-mer peptide modified by the Polyethylene Glycol of the invention can basically maintain the biological activity of the deuterohemin 6-mer peptide, and has better stability than the deuterohemin 6-mer peptide without any modification. The preparation method of the deuterohemin 6-mer peptide modified by the Polyethylene Glycol of the invention is simple and easy to operate, while the structure general formula of the deuterohemin 6-mer peptide modified by the Polyethylene Glycol is shown on the right, wherein, R stands for a DhHP-6 molecule eliminating an NH2.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a heme short peptide compound modified by monomethoxy polyethylene glycol succinimide ester derivatives and a preparation method. Background of the invention [0002] Deuterohemin hexapeptide (Deuterohemin-Ala-His-Thr-Val-Glu-Lys, DhHP-6) is a short peptide containing hypoheme designed based on the structure of microenzyme and natural ascorbate peroxidase , is a better peroxidase mimic. The molecular weight of the short peptidomimetic enzyme is less than 3000, and it can be developed into a high-efficiency antioxidant drug. However, its bioavailability is low and it is susceptible to protease degradation. [0003] Derivatives of monomethoxy polyethylene glycol succinimide ester, a polymer compound with polyethylene glycol as the main chain and a methoxy end group and an activated succinimide ester end group . Among them, polyethylene glycol (PEG) is a hydrophilic, bio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K1/107
Inventor 汤钧林航李惟王丽萍
Owner JILIN UNIV
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