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Method for preparing alpha-small aspartyl-peptide by means of dihydroorotase

A technology of dihydroorotase and aspartyl small peptide, which is applied in the direction of fermentation, etc., can solve the problem that by-products are difficult to remove, and achieve the effects of avoiding protection and deprotection steps, simple operation and single reaction product

Inactive Publication Date: 2014-07-02
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

No matter which synthesis method is used, there will always be a side reaction in which aspartic acid racemization or β-position carboxyl group participates in the synthesis, and the by-product is the isoform formed by the α-carboxyl group of D-Asp or the β-carboxyl group of Asp and amino acid. Constructs, these by-products are difficult to remove

Method used

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  • Method for preparing alpha-small aspartyl-peptide by means of dihydroorotase
  • Method for preparing alpha-small aspartyl-peptide by means of dihydroorotase
  • Method for preparing alpha-small aspartyl-peptide by means of dihydroorotase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Synthesis of dihydroorotoyl-glycine methyl ester.

[0035] Add L-dihydroorotic acid (0.1mol) to 300mL DMF, stir at room temperature to dissolve, add DCC (0.1mol) and HOBt (0.1mol), react for 2h, remove DCU (dicyclohexylurea) by filtration, and take the filtrate. Glycine methyl ester (0.13 mol) was added and reacted overnight. Pour into water, add ethyl acetate to extract three times, the extract is dried and then concentrated to obtain a white solid, which is subjected to silica gel column chromatography, and the eluent is ethyl acetate:chloroform=3:1 (volume ratio), recrystallized from methanol Afterwards, 18.2 g of white crystal dihydroorotoyl-glycine methyl ester was obtained, with a yield of 79.5%; 1 H NMR (DMSO-d 6 , 500MHz) δ: 10.00(s, 1H, 3-NH), 8.46(s, 1H, α-CO-NH), 7.53(s, 1H, 1-NH), 4.04(t, 1H, 6-CH) , 3.86(s, 2H, α-CH 2 ), 3.62 (s, 3H, CH 3 ), 2.58-2.67 (m, 2H, 5-CH 2 ).

Embodiment 2

[0036] Example 2: Synthesis of dihydroorotyl-D-alanine methyl ester.

[0037] Add L-dihydroorotic acid (0.1mol) to 200mL of water, stir at room temperature to dissolve, add DIC (0.12mol) and HOBt (0.12mol), react for 2h, filter to remove the precipitate, and take the filtrate. D-alanine methyl ester (0.12 mol) was added and reacted overnight. Pour into water, add ethyl acetate to extract three times, the extract is dried and concentrated to obtain a white solid, which is subjected to silica gel column chromatography, the eluent is ethyl acetate:chloroform=3:1 (volume ratio), concentrated to obtain a white solid Dihydroorotoyl-D-alanine methyl ester 17.0g, yield 65.5%; 1 H NMR (DMSO-d 6 , 500MHz) δ: 10.14 (s, 1H, 3-NH), 8.78 (s, 1H, α-CO-NH), 7.32 (s, 1H, 1-NH), 4.79 (t, 1H, 6-CH) , 4.18 (m, 1H, α-CH), 3.68 (s, 3H, -OCH 3 ), 2.58-2.67 (m, 2H, 5-CH 2 ), 1.48 (d,3H,β-CH 3 ).

Embodiment 3

[0038] Example 3: Synthesis of dihydroorotoyl-L-phenylalanine methyl ester.

[0039] Add L-dihydroorotic acid (0.05mol) to 100mL water, stir at room temperature to dissolve, add DIC (0.07mol) and HOBt (0.07mol), react for 2h, filter to remove the precipitate, and take the filtrate. Add L-phenylalanine methyl ester (0.07mol) and react overnight. Pour into water, add ethyl acetate to extract three times, the extract is dried and concentrated to obtain a white solid, which is recrystallized from methanol to obtain 14.3 g of white crystal dihydroorotanoyl-L-phenylalanine methyl ester, with a yield of 90.5 %; 1 H NMR (DMSO-d 6 , 500MHz) δ: 9.96 (s, 1H, 3-NH), 8.43 (s, 1H, α-CO-NH), 7.51 (s, 1H, 1-NH), 7.18-7.30 (m, 5H, -C 6 h 5 ), 4.45 (t, 1H, 6-CH), 4.00 (t, 1H, α-CH), 3.62 (s, 3H, -OCH 3 ), 2.91-3.03 (d, 2H, β-CH 2 ), 2.45-2.82 (m, 2H, 5-CH 2 ).

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Abstract

The invention discloses a method for preparing α-aspartyl small peptide by dihydroorotase method. The method includes the following steps: using dihydroorotic acid and amino acid esters or small dipeptide ester compounds to carry out a peptide reaction to form a dihydroorotic acid small peptide ester compound; and then using a dilute alkali solution to hydrolyze the ester bond to expose the Terminal carboxyl group; use dihydroorotase to hydrolyze the dihydroorotate small peptide compound that exposes the corresponding carboxyl group to form an N-carbamylated aspartyl small peptide compound; and then use nitrous acid to hydrolyze the aminomethyl peptide compound. The acyl group can be used to obtain α-aspartyl small peptide compounds. The method of the present invention uses a dihydroorotic acid ring to jointly protect the α-amino group and β-carboxyl group in aspartic acid, exposing only the α-carboxyl group, ensuring the position specificity of the peptide-containing reaction; at the same time, enzymatic hydrolysis is used Ring, which avoids repeated protection and deprotection steps in most chemical synthesis, is simple to operate, and reduces the occurrence of side reactions.

Description

technical field [0001] The invention relates to a method for preparing α-aspartyl small peptide by dihydroorotate enzymatic method. Background technique [0002] [0003] α-Aspartyl small peptides refer to small peptides (generally no more than two amino acids) formed by the α-carboxyl group of aspartic acid and other amino acids or small peptides. These small peptides and Its derivatives have important application value in the fields of food and medicine. Such as various sweeteners: L-α-aspartyl-L-phenylalanine methyl ester (aspartame), L-α-aspartame-N-(2,2,4,4 -Tetramethyl-3-sulfidetrimethylene)-D-alaninamide (alitame), N-[N-(3,3-dimethylbutyl)-L-α-aspartyl] -L-phenylalanine methyl ester (Letian), β-O-benzyl-L-α-aspartyl-L-phenylalanine methyl ester (1000 times sweeter than sucrose), L-α -Aspartyl-O-bornyl-L-serine methyl ester, etc. The sweetness of these sweeteners is more than 200 times that of sucrose, and they are low in calories, pure in taste and high in safe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/02
Inventor 武红丽曹飞张煜薄薇周佳栋姚琴
Owner NANJING TECH UNIV