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Stable isotope-labeled alpha-lactalbumin characteristic peptide segment synthetic method

A technology of stable isotopes and characteristic peptides, applied in the direction of albumin peptides, specific peptides, chemical instruments and methods, etc., can solve the problems of undisclosed synthesis methods of protein characteristic peptides

Inactive Publication Date: 2017-07-04
SHANGHAI RES INST OF CHEM IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the patent discloses a detection method, and does not disclose the synthesis method of protein characteristic peptides

Method used

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preparation example Construction

[0066] A method for synthesizing characteristic peptides of α-lactalbumin labeled with a stable isotope, using a liquid-phase synthetic peptide method, using stable isotope-labeled or non-labeled valine (Val) as the starting material, and Fmoc-protected non-labeled or labeled glycine (Gly ), under the action of a condensing agent, synthesize a dipeptide, and then take off the protecting group Fmoc, and then combine with the non-labeled or labeled isoleucine (Ile) protected by Fmoc to generate a tripeptide under the action of a condensing agent, and take off The protecting group Fmoc, according to this method, is sequentially combined with Fmoc-protected non-marked or labeled asparagine (Asn), tyrosine (Tyr), tryptophan (Trp), leucine (Leu), alanine (Ala ), histidine (His) and Cbz-protected non-labeled or labeled lysine (Lys) to react to finally generate stable isotope labeled α-lactalbumin characteristic peptide VGINYWLAHK, specifically the following steps:

[0067] 1) Stable ...

Embodiment 1

[0091] Stable isotope labeled H-Lys- 13 C 6 -His- 13 C 6 -Ala- 13 C 3 -Leu- 13 C 6 -Trp- 13 C 11 -Tyr- 13 C 9 -Asn- 13 C 4 -Ile- 13 C 6 -Gly- 13 C 2 -Val- 13 C 5 -NH 2 Synthesis

[0092] 1. Fmoc-Gly- 13 C 2 -Val- 13 C 5 -NH 2 Synthesis

[0093] H-Val- 13 C 5 -NH 2 Dissolve 1.22g in 10ml of chloroform, add 1.29g of DIEA under stirring, dissolve and set aside; weigh Fmoc-Gly- 13 C 2 -OH 3.0g and HOCt 3.0g were dissolved in 15ml of chloroform, and DIC 1.5g was added dropwise under stirring, and reacted for 10 minutes, then this solution was added to the initial mixed solution, and reacted at room temperature for 3 hours, and the reaction was complete by TLC , the reaction solution was filtered and washed successively with sodium bicarbonate solution, hydrochloric acid, and saturated brine, then dried with anhydrous magnesium chloride, concentrated under reduced pressure, and crystallized with diethyl ether to obtain 3.45 g of the target product as a ...

Embodiment 2

[0129] Stable isotope labeled H-Lys- 15 N 2 -His- 15 N 3 -Ala- 15 N-Leu- 15 N-Trp- 15 N 2 -Tyr- 15 N-Asn- 15 N-Ile- 15 N-Gly- 15 N-Val- 15 N-NH 2 Synthesis

[0130] 1. Fmoc-Gly- 15 N-Val- 15 N-NH 2 Synthesis

[0131] H-Val- 15 N-NH 2 Dissolve 1.17g in 10ml of chloroform, add 1.29g of DIEA under stirring, dissolve and set aside; weigh Fmoc-Gly- 15 Dissolve 3.0g of N-OH and 3.0g of HOCt in 15ml of chloroform, add 1.5g of DIC dropwise under stirring, react for 10 minutes, then add this solution to the initial mixture, react at room temperature for 3 hours, and detect the reaction by thin-layer chromatography Completely, the reaction solution was filtered and washed successively with sodium bicarbonate solution, hydrochloric acid, and saturated brine, then dried with anhydrous magnesium chloride, concentrated under reduced pressure, crystallized with diethyl ether to obtain 3.48 g of the target product as a white solid, with a yield of 87.7%. 98.9% purity, 99....

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Abstract

The invention relates to a stable isotope-labeled alpha-lactalbumin characteristic peptide segment synthetic method. According to the invention, a liquid-phase polypeptide synthesis method is used, stable isotope-labeled or non-labeled valine is taken as an initial raw material, the initial raw material and Fmoc-protected non-labeled or labeled glycine are subjected to synthesis to obtain dipeptides under effect of a condensing agent, a protective group Fmoc is removed, the material and Fmoc-protected non-labeled or labeled isoleucine are subjected to synthesis to obtain tripeptides under effect of the condensing agent, the tripeptides is reacted with the Fmoc-protected non-labeled or labeled asparagines, tyrosine, tryptophan, leucine, alanine, histidine and Cbz-protected non-labeled or labeled lysine, and finally the stable isotope-labeled alpha-lactalbumin characteristic peptide segment VGINYWLAHK is generated. The chemical purity of the product can reach 99% and above, the isotope abundance is 99% and above, and the product can satisfy the detection requirement of the allergen alpha-lactalbumin in food.

Description

technical field [0001] The invention relates to the field of isotope labeling, in particular to a synthesis method for stable isotope labeling alpha-lactalbumin characteristic peptides. Background technique [0002] Food allergens refer to antigenic substances present in food that can selectively activate human cells, induce specific antibody responses, and cause allergic reactions. Generally, food allergens are proteins or glycoproteins, which belong to different protein families. There are currently about 160 types of food containing allergens that can cause allergic reactions, and about 260 molecules have been labeled as food allergens. Epidemiological studies have shown that 90% of food allergies are caused by 8 types of food, commonly known as the "8 major" allergens, including milk, eggs, soybeans, peanuts, tree nuts, wheat, fish and shellfish. kind. Symptoms of food allergies include hives, vomiting, and asthma, and can be life-threatening in severe cases. Among t...

Claims

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Application Information

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IPC IPC(8): C07K14/76C07K1/06C07K1/02
CPCC07K14/76Y02P20/55
Inventor 徐仲杰罗勇钟佳琪涂亚辉王浩然孙雯卢伟京杨维成
Owner SHANGHAI RES INST OF CHEM IND
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