CN103243141A - Method for preparing gamma-glutamyl small peptide compound by one-pot method - Google Patents
Method for preparing gamma-glutamyl small peptide compound by one-pot method Download PDFInfo
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- CN103243141A CN103243141A CN2013101998665A CN201310199866A CN103243141A CN 103243141 A CN103243141 A CN 103243141A CN 2013101998665 A CN2013101998665 A CN 2013101998665A CN 201310199866 A CN201310199866 A CN 201310199866A CN 103243141 A CN103243141 A CN 103243141A
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- Prior art keywords
- glutamyl
- gamma
- small peptide
- glycolylurea
- peptide compound
- Prior art date
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Landscapes
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Abstract
The invention discloses a method for preparing gamma-glutamyl small peptide compounds by a one-pot method. The method comprises the following steps: 5-carboxyethyl hydantoin small peptide compounds are formed by carrying out water-phase peptide grafting reaction on 5-carboxyethyl hydantoin and amino acid; directly adding thallus containing hydantoinase and carbamoylase as a catalyst to perform enzyme catalytic hydrolysis ring-opening reaction without extraction and separation after the reaction is finished, thereby obtaining the gamma-glutamyl small peptide compound. The method adopts hydantoin rings to protect alpha-amino and alpha-carboxyl of glutamic acid together, only gamma-carboxyl is exposed, and the position specificity of the peptide-joining reaction is ensured; the water phase peptide is adopted, the intermediate product is directly subjected to the next enzyme reaction without extraction and separation, the total reaction yield can be effectively improved in the one-pot reaction process, and the problem of environmental pollution caused by an organic solvent is avoided in the water phase reaction; the enzymatic hydrolysis ring opening is adopted, so that repeated protection and deprotection steps in most chemical synthesis are avoided, and side reactions are reduced.
Description
Technical field
The present invention relates to a kind of method that adopts one kettle way to prepare gamma-glutamyl small peptide compound.
Background technology
Gamma-glutamyl small peptide compound (1) is the small peptide compound (generally being no more than two amino acid) that the alpha-amino group by the γ-carboxyl of L-glutamic acid and other amino acid (or amino acid derivative) is connected to form by γ-peptide bond.Gamma-glutamyl small peptide compound is difficult for by general protease hydrolysis in vivo owing to have special γ-peptide bond structure, thus this compounds wide participation some important metabolic processes in the organism.As the important amino acid derived compound of a class, gamma-glutamyl small peptide compound has been widely used in fields such as food, medicine, makeup, fodder additives.Nearest studies show that, the synthetic precursor of gsh---gamma-L-glutamine-cysteine has the physiological function similar to gsh, and can effectively realize striding the film transportation, improve born of the same parents' glutathion inside horizontal aspect, its function is better than and directly supplies with gsh outside born of the same parents, be a kind of anti-aging product that has market outlook (John B W, Hani Z M.Process for the Producyion γ-Glutamylcysteine.AU:2006228996,2006-10-05.).Simultaneously, the derivative of one class gamma-D-glutamine acyl L-tryptophane or γ-L-glutamy-L-tryptophane also is proved to be has significant immunoregulation effect (Suzuki H, Kato K, Kumagai H.Development of an Efficient Enzymatic Production of γ-D-Glutamyl-L-tryptophan (SCV-07), a Prospective Medicine for Tuberculosis, with Bacterial γ-Glutamyltranspeptidase.J.Biotechnol., 2004,111:291～295.; Kolobov A A, Simbirtsev A S, Kulikov S V, et al. γ-L-Glutamyl Containing Immunomodulator Compounds and Methods Therewith.US:5744452,1998-04-28.).In addition; the research that gamma-glutamylization is applied to develop the novel targeted medicine (as γ-D-glutamy glycine) at liver, courage, renal tissue and nervous system disorders has also caused concern (John B W widely; Hani Z M.Process for the Producyion γ-Glutamylcysteine.AU:2006228996,2006-10-05.).
At present, the synthetic method of gamma-glutamyl small peptide compound mainly contains extraction method, fermentation method, chemical synthesis and enzyme process.Extraction method is classical production method, be raw material with animal and plant tissue and the yeast that contains gamma-glutamyl small peptide compound mainly, by the interpolation appropriate solvent or in conjunction with amylase, protease treatment, separation and purification forms again, but because the content of gamma-glutamyl small peptide compound is all very low in the raw material, need to consume shortcomings such as a large amount of raw materials and solvent, therefore seldom use.Fermentative Production is exactly to cultivate prescription, set up and optimize fermentation control strategy, improvement and raising downstream process technology etc. by the synthetic ability of seed selection (structure) gamma-glutamyl small peptide compound microorganism, screening and optimization strong and that intracellular product content is high, final yield and the quality that improves product, the subject matter of fermentation method is that transformation efficiency is low, fermentation period is long, cross low product content and need complicated separating technology, equipment has high input, the production cost height, and product purity and yield are relatively low, thereby have limited the further popularization of this method.Chemical synthesis all needs usually to the alpha-amino group of L-glutamic acid and the proceed step by step protection respectively of α-carboxyl; γ-the carboxyl that only dissociates connects reactive polypeptide; connecing also needs substep respectively that the protecting group of alpha-amino group and α-carboxyl is removed again after peptide finishes; like this in repeated protection and deprotection process; cause phenomenons such as peptide bond rupture, racemization easily, thereby make that yield reduces, optical purity reduces.Suzuki etc. utilize gamma glutamyl transpeptidase, carried out comparatively careful research for the enzyme process of gamma-glutamyl small peptide compound is synthetic, prepared γ-L-glutamy-L-Histidine, γ-L-glutamy-L-L-Tyrosine methyl ester, a series of gamma-glutamyl small peptide compounds (Suzuki H such as γ-L-glutamy-L-phenylalanine and gamma-D-glutamine acyl L-tryptophane, Yamada C, Kato K. γ-Glutamyl Compounds and Their Enzymatic Production Using Bacterial γ-Glutamyltranspeptidase.Amino Acids, 2007,32:333～340.), but because having concurrently, gamma glutamyl transpeptidase changes peptide and hydrolytic activity, in reaction process, often be accompanied by side reactions such as rotation peptide and product hydrolysis, make reaction process be difficult to control, had a strong impact on reaction efficiency.We before research (Cao Fei, Zhou Jiadong, Wang Yuexia, Zhang Furong, Huang Rongxing, Wei Ping. a kind of method .CN:ZL200910213531.8 of preparing glutathione using hydantoinase, 2009-11-05.; Cao Fei, Wei Ping, Zhou Jiadong, Wang Yuexia, Wu Hongli, Huang Rong wakes up, Zhang Furong. a kind of method .CN:ZL200910183579.9 of preparing optically active theanine by enzymatic method, 2009-09-23.) in, using hydantoinase is applied to the synthetic of gamma-glutamyl compounds such as gsh and theanine, but also there are following two aspect problems: on the one hand, carrying out gamma-glutamyl when connecing reactive polypeptide, employing be that organic peptide reagent that joins carries out in organic solvent, and organic solvent is unfriendly to environment, human body there is harm, the general boiling point of organic solvent is higher simultaneously, needs higher cost when product separates; On the other hand, the product that connects reactive polypeptide needs at first from organic solvent separation and Extraction to come out, and then joins aqueous phase and carry out the enzymatic hydrolysis ring-opening reaction, and leaching process can cause the portion of product loss, causes total recovery to descend.
Summary of the invention
Technical problem to be solved by this invention provide a kind of easy, do not produce racemization and alpha-position carboxyl participate in reaction, intermediate product need not extraction separation, water " one kettle way " prepares the method for gamma-glutamyl small peptide compound.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
The present invention is initiator with 5-propyloic glycolylurea; 5-propyloic glycolylurea its structure itself protects altogether with the form of glycolylurea ring alpha-amino group and the α-carboxyl with L-glutamic acid just; only expose γ-carboxyl; guaranteed to connect the position specific of reactive polypeptide; introducing water simultaneously connects peptide reagent connect reactive polypeptide in water; reaction need not extraction separation after finishing, and can directly to add the thalline that contains glycolylurea enzyme and carbamoylase simultaneously be that catalyzer carries out next step enzymatic hydrolysis ring-opening reaction; the common protection of one-step removal alpha-amino group and α-carboxyl obtains gamma-glutamyl small peptide compound.What this " one kettle way " reaction process had reduced L-glutamic acid adds steps such as protecting group, deprotection base; do not produce the problem that racemization and alpha-position carboxyl participate in reaction; reaction product is single; intermediate product need not extraction separation can directly carry out next step reaction; effectively improved overall yield of reaction, water react has been avoided the problem of environmental pollution that organic solvent brings in addition.
A kind of one kettle way prepares the method for gamma-glutamyl small peptide compound, comprises the following steps:
(1) 5-propyloic glycolylurea and amino acid carry out water and connect reactive polypeptide and prepare 5-propyloic glycolylurea small peptide compound.
In water, adopt water to connect peptide reagent 5-propyloic glycolylurea (compound 2) terminal carboxyl(group) is connected amino acid (compound 3), obtain 5-propyloic glycolylurea small peptide compound (compound 4).The mol ratio that water connects peptide reagent and 5-propyloic glycolylurea is 1～1.4: 1, and the mol ratio of amino acid and 5-propyloic glycolylurea is 1～1.6: 1,5-propyloic glycolylurea small peptide compound need not separation and Extraction and directly carries out next step enzymatic hydrolysis ring-opening reaction.
Wherein, adopt water to connect peptide reagent and connect amino acid at 5-propyloic glycolylurea terminal carboxyl(group), concrete grammar adds amino acid reaction 10～18h again for earlier water being met peptide reagent and 5-propyloic glycolylurea reaction 1～8h.It is EDCHCl/HOBt (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride/1-hydroxyl-benzotriazole), EDCHCl/HOOBt (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride/3-hydroxyl-1 that described water connects peptide reagent, 2,3-phentriazine-4 (3H)-ketone), any one among EDCHCl/HOAt (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride/1-hydroxyl-7-azo benzotriazole) and the EDCHCl/HOSu (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride/N-hydroxy-succinamide).The R group is any one of amino-acid residue; amino acid comprises glycine; L-amino acid (L-L-Ala; the L-Xie Ansuan; the L-leucine; the L-Isoleucine; the L-Serine; the L-Threonine; the L-halfcystine; the L-methionine(Met); the L-aspartic acid; L-L-glutamic acid; altheine; L-glutaminate; L-Methionin; the L-arginine; the L-phenylalanine; L-tyrosine; the L-tryptophane; the L-Histidine; L-proline(Pro) etc.); D-amino acid (D-L-Ala; the D-Xie Ansuan; the D-leucine; the D-Isoleucine; the D-Serine; the D-Threonine; the D-halfcystine; the D-methionine(Met); the D-aspartic acid; D-L-glutamic acid; the D-l-asparagine; the D-glutamine; D-Methionin; the D-arginine; the D-phenylalanine; D-tyrosine; the D-tryptophane; the D-Histidine; D-proline(Pro) etc.), side chain protected L-amino acid (O-protection-L-Serine; O-protection-L-Threonine; S-protection-L-halfcystine; N
ε-protection-L-Methionin, N
ω-protection-L-arginine, O-protection-L-tyrosine, N
Im-protection-L-Histidine etc.) and side chain protected D-amino acid (O-protection-D-Serine, O-protection-D-Threonine, S-protection-D-halfcystine, N
ε-protection-D-Methionin, N
ω-protection-D-arginine, O-protection-D-tyrosine, N
Im-protection-D-Histidine etc.).Wherein, side chain protected L-amino acid and the amino acid whose protecting group of side chain protected D-are protecting group commonly used (Sewald N, the Jakubke H-D.Peptides:Chemistry and Biology.Weinheim:WILEY-VCH Verlag GmbH﹠amp during general polypeptide synthesizes; Co.KGaA, 2002.; Huang Weide, Chen Changqing. polypeptide is synthetic. Beijing: Science Press, 1985.; Green T W, Wuts P G M.Protective Group in Organic Synthesis.Third Edition.New York:John Wiley﹠amp; Son, Inc., 1999.).
(2) 5-propyloic glycolylurea small peptide compound carries out the enzymatic hydrolysis ring-opening reaction and prepares gamma-glutamyl small peptide compound.
Be catalyzer with the thalline that contains glycolylurea enzyme and carbamoylase simultaneously, under weak basic condition, catalytic hydrolysis 5-propyloic glycolylurea small peptide compound (compound 4) open loop prepares corresponding gamma-glutamyl small peptide compound (compound 1), with the enzymatic conversion solution separating of gained, the concentrated gamma-glutamyl small peptide compound that obtains.
Wherein, the described thalline that contains glycolylurea enzyme and carbamoylase simultaneously is genus bacillus MH602 (Bacillus fordii MH602, China's typical culture collection center preservation, CCTCC NO.M206144) or onion bulkholderia cepasea JS-02 (Burkholderia cepecia JS-02, China's typical culture collection center preservation, CCTCC NO.M202047).Being that catalyzer can prepare γ-L-glutamy small peptide compound with genus bacillus MH602, is that catalyzer can prepare γ-D-glutamy small peptide compound with onion bulkholderia cepasea JS-02.Described weak basic condition is pH7.5～9.5.The weight ratio of thalline and 5-propyloic glycolylurea small peptide compound is 1: 0.5～8.Catalytic hydrolysis ring-opening reaction temperature is 35～45 ℃, and the reaction times is 10～24h.
(3) deprotection of the gamma-glutamyl small peptide compound of part side chain band protecting group.
The gamma-glutamyl small peptide compound of the part side chain band protecting group that step (2) is obtained is (as gamma-glutamyl-O-protection-Serine, gamma-glutamyl-O-protection-Threonine, gamma-glutamyl-S-protection-halfcystine, gamma-glutamyl-N
ε-protection-Methionin, gamma-glutamyl-N
ω-protection-arginine, gamma-glutamyl-O-protection-tyrosine, gamma-glutamyl-N
Im-protection-Histidine etc.) remove protecting group, after filtration (or extraction), concentrate, ion-exchange separates the gamma-glutamyl small peptide compound that obtains side chain unprotect base.
Wherein, described Side chain protective group R
1, R
2, R
3, R
4, R
5, R
6, R
7Be the protecting group commonly used during generally polypeptide synthesizes, the described method that removes protecting group is conventional deprotection method (Sewald N, the Jakubke H-D.Peptides:Chemistry and Biology.Weinheim:WILEY-VCH Verlag GmbH﹠amp during general polypeptide synthesizes; Co.KGaA, 2002.; Huang Weide, Chen Changqing. polypeptide is synthetic. Beijing: Science Press, 1985.; Green T W, Wuts P G M.Protective Group in Organic Synthesis.Third Edition.New York:John Wiley﹠amp; Son; Inc.; 1999.); as adopt the Pd/C catalyst hydrogenation to remove S-protecting group such as S-benzyl in gamma-glutamyl-S-protection-halfcystine; hydrogenation conditions is 20～60 ℃, stirring reaction 1～10h; the Pd/C catalyzer is 0.5%～20%Pd/C catalyzer, and the hydrogenation reaction hydrogen donor is any one in formic acid and salt, hydrazine, Hypophosporous Acid, 50 and salt thereof, tetrahydrobenzene and the hydrogen.
Beneficial effect of the present invention:
One kettle way of the present invention prepares gamma-glutamyl small peptide compound method and has compared following advantage with other preparation methods:
1, employing glycolylurea ring is protected alpha-amino group and the α-carboxyl in the L-glutamic acid altogether, only exposes γ-carboxyl, has guaranteed to connect the position specific of reactive polypeptide.
2, adopt water to connect peptide reagent and in water, connect reactive polypeptide, intermediate product need not extraction separation can directly carry out next step enzymatic hydrolysis ring-opening reaction, " one kettle way " effectively improved overall yield of reaction, and water react has been avoided the problem of environmental pollution that organic solvent brings in addition.
3, adopting the thalline that contains glycolylurea enzyme and carbamoylase simultaneously is the open loop of catalyst hydrolysis glycolylurea ring; the common protection of one-step removal alpha-amino group and α-carboxyl; obtain gamma-glutamyl small peptide compound, avoided repeated protection and deprotection steps in most chemosynthesis, simple to operate.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand that the described concrete material proportion of embodiment, reaction conditions and result thereof only are used for explanation the present invention, and should also can not limit the present invention described in detail in claims.
Embodiment 1:5-propyloic glycolylurea and S-benzyl-L-halfcystine carry out water and connect reactive polypeptide and prepare N-glycolylurea propionyl-S-benzyl-L-halfcystine.
5-propyloic glycolylurea (0.01mol) adds in the 25mL water, the stirring at room dissolving, add EDCHCl (0.01mol) and HOOBt (0.01mol), reaction 2h, add S-benzyl-L-halfcystine (0.013mol), reaction is spent the night, and obtains N-glycolylurea propionyl-S-benzyl-L-halfcystine aqueous solution, need not extraction separation and directly carries out next step enzymatic hydrolysis ring-opening reaction.Sampling is carried out product and is characterized: yield 95%,
1H NMR (DMSO-d
6, 500MHz) δ: 12.80 (s, 1H), 10.62 (s, 1H), 8.25 (d, J=8.0Hz, 1H), 7.89 (s, 1H), 7.32～7.24 (m, 5H), 4.45～4.41 (m, 1H), 4.05～4.03 (m, 1H), 3.75 (s, 2H), 2.81～2.63 (m, 2H), 2.31～2.17 (m, 2H), 1.96～1.70 (m, 2H); HRMS (ESI) calcd for C
16H
19N
3NaO
5S[M+Na]
+388.0938, found388.0946.(reactions steps: 5-propyloic glycolylurea (0.01mol) adds 25mL DMF to contrast organic reactive polypeptide that joins, the stirring at room dissolving, add DIC (0.01mol) and HOOBt (0.01mol), reaction 2h crosses leaching filtrate, add S-benzyl-L-halfcystine (0.013mol), reaction is spent the night, and pours in the water, obtains N-glycolylurea propionyl-S-benzyl-L-halfcystine 3.3g behind the extraction condensing crystal, yield 86%), yield is lower than water and connects reactive polypeptide.
Embodiment 2:5-propyloic glycolylurea and O-benzyl-D-Serine carry out water and connect reactive polypeptide and prepare N-glycolylurea propionyl-O-benzyl-D-Serine.
5-propyloic glycolylurea (0.01mol) adds 25mL water, the stirring at room dissolving, add EDCHCl (0.011mol) and HOAt (0.011mol), reaction 6h, add O-benzyl-D-Serine (0.012mol), reaction is spent the night, and obtains N-glycolylurea propionyl-O-benzyl-D-Serine aqueous solution, need not extraction separation and directly carries out next step enzymatic hydrolysis ring-opening reaction.Sampling is carried out product and is characterized: yield 96%,
1H NMR (DMSO-d
6, 500MHz) δ: 12.78 (s, 1H), 10.62 (s, 1H), 8.24 (d, J=8.0Hz, 1H), 7.88 (s, 1H), 7.34～7.26 (m, 5H), 4.62～4.56 (m, 1H), 4.51 (s, 2H), 4.03～3.99 (m, 1H), 3.74 (dd, J=9.7,5.8Hz, 1H), 3.63 (dd, J=9.7,4.2Hz, 1H), 2.32～2.21 (m, 2H), 1.94～1.72 (m, 2H); HRMS (ESI) calcd for C
16H
19N
3NaO
6[M+Na]
+372.1166, found372.1162.(reactions steps: 5-propyloic glycolylurea (0.01mol) adds 25mL DMF to contrast organic reactive polypeptide that joins, the stirring at room dissolving, add DIC (0.011mol) and HOAt (0.011mol), reaction 6h crosses leaching filtrate, add O-benzyl-D-Serine (0.012mol), reaction is spent the night, and pours in the water, obtains N-glycolylurea propionyl-O-benzyl-D-Serine 3.3g behind the extraction condensing crystal, yield 89%), yield is lower than water and connects reactive polypeptide.
Embodiment 3:5-propyloic glycolylurea and L-tryptophane carry out water and connect reactive polypeptide and prepare N-glycolylurea propionyl-L-tryptophane.
5-propyloic glycolylurea (0.01mol) adds 25mL water, the ice-water bath stirring and dissolving, add EDCHCl (0.012mol) and HOBt (0.012mol), reaction 8h, add L-tryptophane (0.015mol), reaction is spent the night, and obtains N-glycolylurea propionyl-L-tryptophane aqueous solution, need not extraction separation and directly carries out next step enzymatic hydrolysis ring-opening reaction.Sampling is carried out product and is characterized: yield 93%, MS m/z:357.4 (M-1)
-(reactions steps: 5-propyloic glycolylurea (0.01mol) adds 25mL DMF to contrast organic reactive polypeptide that joins, the ice-water bath stirring and dissolving, add DCC (0.012mol) and HOBt (0.012mol), reaction 8h crosses leaching filtrate, add L-tryptophane (0.015mol), reaction is spent the night, and pours in the water, obtains N-glycolylurea propionyl-L-tryptophane 3.1g behind the extraction condensing crystal, yield 87%), yield is lower than water and connects reactive polypeptide.
Embodiment 4:5-propyloic glycolylurea and L-L-glutamic acid carry out water and connect reactive polypeptide and prepare N-glycolylurea propionyl-L-L-glutamic acid.
Adopt and embodiment 3 similar methods, changing the L-tryptophane is L-L-glutamic acid, obtains N-glycolylurea propionyl-L-glutamic acid aqueous solution, and sampling is carried out product and characterized: yield 94%, MS m/z:300.2 (M-1)
-Contrast organic reactive polypeptide that joins (reactions steps is similar to embodiment 3, and changing the L-tryptophane is L-L-glutamic acid, obtains N-glycolylurea propionyl-L-L-glutamic acid 2.6g, yield 87%), yield is lower than water and connects reactive polypeptide.
Embodiment 5:5-propyloic glycolylurea and D-L-Ala carry out water and connect reactive polypeptide and prepare N-glycolylurea propionyl-D-L-Ala.
Adopt and embodiment 3 similar methods, changing the L-tryptophane is the D-L-Ala, obtains N-glycolylurea propionyl-D-L-Ala aqueous solution, and sampling is carried out product and characterized: yield 91%, MS m/z:241.9 (M-1)
-Contrast organic reactive polypeptide that joins (reactions steps is similar to embodiment 3, and changing the L-tryptophane is the D-L-Ala, obtains N-glycolylurea propionyl-D-L-Ala 2.0g, yield 83%), yield is lower than water and connects reactive polypeptide.
Embodiment 6:5-propyloic glycolylurea and L-phenylalanine carry out water and connect reactive polypeptide and prepare N-glycolylurea propionyl-L-phenylalanine.
Adopt and embodiment 3 similar methods, changing the L-tryptophane is the L-phenylalanine, obtains N-glycolylurea propionyl-L-phenylalanine aqueous solution, and sampling is carried out product and characterized: yield 92%, MS m/z:318.3 (M-1)
-Contrast organic reactive polypeptide that joins (reactions steps is similar to embodiment 3, and changing the L-tryptophane is the L-phenylalanine, obtains N-glycolylurea propionyl-L-phenylalanine 2.7g, yield 85%), yield is lower than water and connects reactive polypeptide.
Embodiment 7: enzymatic production I.
Get glucose 20g, corn steep liquor 20mL, K
2HPO
415g, MgSO
40.5g, 5-propyloic glycolylurea 5g, pH7.5 adds tap water and is mixed with the 0.5L liquid fermentation medium, and packing 50mL substratum adds 8 layers of gauze to 500mL, and after wrapping with kraft paper, 25min is standby for the 0.1MPa high pressure steam sterilization.
With genus bacillus MH602 (Bacillus fordii MH602, CCTCC NO.M206144), be connected on fresh slant medium (slant medium (gL
-1: peptone 10, extractum carnis 3, NaCl5, agar 20) on, activation culture 24h inserts seed culture medium (seed culture medium (gL
-1): glucose 15, peptone 20, K
2HPO
42, MgSO
40.25) at 33 ℃, 220rmin
-1Shaking culture 20h cultivates and finishes to insert fermention medium, in 33 ℃, 200rmin
-1Fermentation culture 24h, after the fermentation ends, 8000rmin
-1Centrifugal 25min collects wet thallus.
Embodiment 8: enzymatic production II.
Get starch 20g, peptone 20g, K
2HPO
415g, MgSO
40.5g, 5-methylthio ethyl glycolylurea 5g, pH7.5 adds tap water and is mixed with the 0.5L liquid fermentation medium, and packing 50mL substratum adds 8 layers of gauze to 500mL, and after wrapping with kraft paper, 25min is standby for the 0.1MPa high pressure steam sterilization.
With onion bulkholderia cepasea JS-02 (Burkholderia cepecia JS-02, CCTCC NO.M202047), be connected on fresh slant medium (slant medium (gL
-1): peptone 10, extractum carnis 3, NaCl5, agar 20) on, activation culture 24h inserts seed culture medium (seed culture medium (gL
-1): glucose 15, peptone 20, K
2HPO
42, MgSO
40.25) at 33 ℃, 220rmin
-1Shaking culture 20h.Cultivate and finish to insert fermention medium, in 33 ℃, 200rmin
-1Fermentation culture 24h.After the fermentation ends, 8000rmin
-1Centrifugal 25min collects wet thallus.
Embodiment 9: enzymatic hydrolysis N-glycolylurea propionyl-S-benzyl-L-halfcystine prepares γ-L-glutamy-S-benzyl-L-halfcystine.
That gets that embodiment 1 obtains obtains N-glycolylurea propionyl-S-benzyl-L-halfcystine aqueous solution, adds the Tris-HCl aqueous solution (pH9.0,0.1molL
-1) be diluted to 100mL, add the wet thallus 2g that embodiment 7 obtains, mix and place shaking bath reaction 18h, temperature of reaction is 37 ℃.After reaction finishes, 8000rmin
-1Centrifugal 15min removes thalline, goes in the anion-exchange column, obtains γ-L-glutamy-S-benzyl-L-halfcystine 2.9g behind the wash-out condensing crystal, yield 85%, [α]
D 25-5.5 (c0.10, H
2O);
1H NMR (D
2O, 500MHz) δ: 7.43～7.34 (m, 5H), 4.33 (dd, J=8.1,4.4Hz, 1H), 3.81 (s, 2H), 3.76 (t, J=6.3Hz, 1H), 2.97 (dd, J=13.9,4.4Hz, 1H), 2.83 (dd, J=13.9,8.2Hz, 1H), 2.45 (t, J=7.6Hz, 2H), 2.20～2.06 (m, 2H); HRMS (ESI) calcd for C
15H
20N
2NaO
5S[M+Na]
+363.0985, found363.0996.
Embodiment 10: γ-L-glutamy-S-benzyl-L-halfcystine deprotection base prepares gamma-L-glutamine-cysteine.
Get γ-L-glutamy-S-benzyl-L-halfcystine 1.0g that embodiment 9 obtains, add among the 20mL MeOH, add 2.0g10%Pd/C catalyzer and 0.5g ammonium formiate (hydrogen donor), 50 ℃ of reaction 10h, cooled and filtered, with MeOH washing Pd/C catalyzer twice, merging filtrate concentrates, go in the anion-exchange column, behind the wash-out condensing crystal, obtain gamma-L-glutamine-cysteine 0.65g, yield 89%, [α]
D 25+ 13.4 (c0.10, H
2O);
1H NMR (D
2O, 500MHz) δ: 4.58 (t, J=6.0Hz, 1H), 3.51 (t, J=6.3Hz, 1H), 2.92～2.88 (m, 2H), 2.51～2.42 (m, 2H), 2.19～2.11 (m, 2H); HRMS (ESI) calcd for C
8H
14N
2NaO
5S[M+Na]
+273.0515, found273.0624.
Embodiment 11: enzymatic hydrolysis N-glycolylurea propionyl-O-benzyl-D-Serine prepares γ-D-glutamy-O-benzyl-D-Serine.
That gets that embodiment 2 obtains obtains N-glycolylurea propionyl-O-benzyl-D-Serine aqueous solution, adds the Tris-HCl aqueous solution (pH8.5,0.05molL
-1) be diluted to 100mL, add the wet thallus 1.5g that embodiment 8 obtains, mix and place shaking bath reaction 20h, temperature of reaction is 37 ℃.After reaction finishes, 8000rmin
-1Centrifugal 15min removes thalline, goes in the anion-exchange column, obtains γ-D-glutamy-O-benzyl-D-Serine 3.0g behind the wash-out condensing crystal, yield 87%, [α]
D 25+ 22.1 (c0.10, H
2O);
1H NMR (D
2O, 500MHz) δ: 7.41～7.33 (m, 5H), 4.61 (s, 2H), 4.43 (dd, J=8.2,4.9Hz, 1H), 3.95 (dd, J=14.1,4.9Hz, 1H), 3.84 (dd, J=14.1,8.3Hz, 1H), 3.73 (t, J=6.1Hz, 1H), 2.48 (t, J=7.4Hz, 2H), 2.19～2.07 (m, 2H); HRMS (ESI) calcd for C
15H
20N
2NaO
6[M+Na]
+347.1213, found347.1223.
Embodiment 12: γ-D-glutamy-O-benzyl-D-Serine deprotection base prepares γ-D-glutamy-D-Serine.
Get γ-D-glutamy-O-benzyl-D-Serine 2.0g that embodiment 11 obtains, add among the 50mL MeOH, add the 3.0g15%Pd/C catalyzer, the compression steel cylinder feeds hydrogen (hydrogen donor), 40 ℃ of reaction 8h, cooled and filtered is washed the Pd/C catalyzer twice with MeOH, merging filtrate, concentrate, go in the anion-exchange column, behind the wash-out condensing crystal, obtain γ-D-glutamy-D-Serine 1.3g, yield 91%, [α]
D 25+ 32.4 (c0.10, H
2O);
1H NMR (D
2O, 500MHz) δ: 4.55 (t, J=5.9Hz, 1H), 4.12～3.87 (m, 2H), 3.49 (t, J=6.1Hz, 1H), 2.49～2.41 (m, 2H), 2.18～2.10 (m, 2H); HRMS (ESI) calcd for C
8H
14N
2NaO
6[M+Na]
+257.0744, found257.0756.
Embodiment 13: enzymatic hydrolysis N-glycolylurea propionyl-L-tryptophane prepares γ-L-glutamy-L-tryptophane.
Adopt and embodiment 9 similar methods, change N-glycolylurea propionyl-L-tryptophane aqueous solution that N-glycolylurea propionyl-S-benzyl-the L-halfcystine aqueous solution obtains for embodiment 3, obtain γ-L-glutamy-L-tryptophane 2.7g, yield 88%, [α]
D 25+ 10.9 (c0.10, H
2O);
1H NMR (D
2O, 500MHz) δ: 7.69～7.16 (m, 5H), 4.59 (dd, J=9.1,5.0Hz, 1H), 3.55 (t, J=6.0Hz, 1H), 3.34 (dd, J=14.4,5.1Hz, 1H), 3.13 (dd, J=14.4,9.0Hz, 1H), 2.32～2.25 (m, 2H), 1.94～1.88 (m, 2H); HRMS (ESI) calcd for C
16H
19N
3NaO
5[M+Na]
+356.1217, found356.1226.
Embodiment 14: enzymatic hydrolysis N-glycolylurea propionyl-L-L-glutamic acid prepares γ-L-glutamy-L-L-glutamic acid.
Adopt and embodiment 9 similar methods, what change that N-glycolylurea propionyl-S-benzyl-L-halfcystine aqueous solution obtains for embodiment 4 obtains N-glycolylurea propionyl-L-glutamic acid aqueous solution, obtains γ-L-glutamy-L-L-glutamic acid 2.2g, yield 86%, [α]
D 25+ 5.4 (c0.10, H
2O);
1H NMR (D
2O, 500MHz) δ: 4.14 (dd, J=9.0,4.8Hz, 1H), 3.71 (t, J=6.2Hz, 1H), 2.45～2.25 (m, 4H), 2.15～1.81 (m, 4H); HRMS (ESI) calcd for C
10H
16N
2NaO
7[M+Na]
+299.0849, found299.0862.
Embodiment 15: enzymatic hydrolysis N-glycolylurea propionyl-D-L-Ala prepares γ-L-glutamy-D-L-Ala.
Adopt and embodiment 9 similar methods, what change that N-glycolylurea propionyl-S-benzyl-L-halfcystine aqueous solution obtains for embodiment 5 obtains N-glycolylurea propionyl-D-L-Ala aqueous solution, obtains γ-L-glutamy-D-L-Ala 1.6g, yield 82%, [α]
D 25+ 45.2 (c0.10, H
2O);
1H NMR (D
2O, 500MHz) δ: 4.11 (q, J=7.3Hz, 1H), 3.38 (t, J=5.5Hz, 1H), 2.38～2.31 (m, 2H), 1.96～1.81 (m, 2H), 1.37 (d, J=7.3Hz, 3H); HRMS (ESI) calcd forC
8H
14N
2NaO
5[M+Na]
+241.0795, found241.0802.
Embodiment 16: enzymatic hydrolysis N-glycolylurea propionyl-L-phenylalanine prepares γ-D-glutamy-L-phenylalanine.
Adopt and embodiment 11 similar methods, what change that N-glycolylurea propionyl-O-benzyl-D-Serine aqueous solution obtains for embodiment 6 obtains N-glycolylurea propionyl-L-phenylalanine aqueous solution, obtains γ-D-glutamy-L-phenylalanine 2.5g, yield 91%, [α]
D 25+ 4.4 (c0.10, H
2O);
1H NMR (D
2O, 500MHz) δ: 7.34～7.23 (m, 5H), 4.44 (dd, J=8.9,5.0Hz, 1H), 3.62 (t, J=6.1Hz, 1H), 3.24 (dd, J=14.1,5.0Hz, 1H), 2.92 (dd, J=14.2,9.0Hz, 1H), 2.38～2.28 (m, 2H), 2.07～1.95 (m, 2H); HRMS (ESI) calcd for C
14H
18N
2NaO
5[M+Na]
+317.1108, found317.1107.
Claims (10)
1. an one kettle way prepares the method for gamma-glutamyl small peptide compound (1), it is characterized in that this method comprises the following steps:
1) is initiator with 5-propyloic glycolylurea, connects at water with amino acid and carry out water under the peptide reagent effect and connect reactive polypeptide, form 5-propyloic glycolylurea small peptide compound, need not extraction separation and directly carry out next step enzyme reaction;
2) the 5-propyloic glycolylurea small peptide compound that obtains of step 1) under weak basic condition, is catalyzer with the thalline that contains glycolylurea enzyme and carbamoylase simultaneously, and the catalytic hydrolysis open loop obtains corresponding gamma-glutamyl small peptide compound.
2. one kettle way according to claim 1 prepares the method for gamma-glutamyl small peptide compound, and the R group that it is characterized in that described gamma-glutamyl small peptide compound (1) is glycine, L-amino acid, D-amino acid, side chain protected L-amino acid or the amino acid whose residue of side chain protected D-.
3. one kettle way according to claim 1 prepares the method for gamma-glutamyl small peptide compound, it is characterized in that in the step 1) that it is among EDCHCl/HOBt, EDCHCl/HOOBt, EDCHCl/HOAt and the EDCHCl/HOSu any one that described water connects peptide reagent.
4. one kettle way according to claim 1 prepares the method for gamma-glutamyl small peptide compound, it is characterized in that in the step 1), the mol ratio that described water connects peptide reagent and 5-propyloic glycolylurea is 1～1.4: 1, and the mol ratio of amino acid and 5-propyloic glycolylurea is 1～1.6: 1.
5. one kettle way according to claim 1 prepares the method for gamma-glutamyl small peptide compound, it is characterized in that in the step 1), described water connects reactive polypeptide for earlier water being met peptide reagent and 5-propyloic glycolylurea reaction 1～8h, adds amino acid reaction 10～18h again.
6. one kettle way according to claim 1 prepares the method for gamma-glutamyl small peptide compound, it is characterized in that step 2) in, the described thalline that contains glycolylurea enzyme and carbamoylase simultaneously is genus bacillus (Bacillus fordii) MH602 or onion bulkholderia cepasea (Burkholderia cepecia) JS-02.
7. one kettle way according to claim 1 prepares the method for gamma-glutamyl small peptide compound, it is characterized in that step 2) in, described weak basic condition is pH7.5～9.5.
8. one kettle way according to claim 1 prepares the method for gamma-glutamyl small peptide compound, it is characterized in that step 2) in, the weight ratio of described thalline and 5-propyloic glycolylurea small peptide compound is 1: 0.5～8.
9. one kettle way according to claim 1 prepares the method for gamma-glutamyl small peptide compound, it is characterized in that step 2) in, described catalytic hydrolysis ring-opening reaction temperature is 35～45 ℃, the reaction times is 10～24h.
10. one kettle way according to claim 2 prepares the method for gamma-glutamyl small peptide compound, it is characterized in that described L-amino acid is any one in L-L-Ala, L-Xie Ansuan, L-leucine, L-Isoleucine, L-Serine, L-Threonine, L-halfcystine, L-methionine(Met), L-aspartic acid, L-L-glutamic acid, altheine, L-glutaminate, L-Methionin, L-arginine, L-phenylalanine, L-tyrosine, L-tryptophane, L-Histidine and the L-proline(Pro);
Described D-amino acid is any one in D-L-Ala, D-Xie Ansuan, D-leucine, D-Isoleucine, D-Serine, D-Threonine, D-halfcystine, D-methionine(Met), D-aspartic acid, D-L-glutamic acid, D-l-asparagine, D-glutamine, D-Methionin, D-arginine, D-phenylalanine, D-tyrosine, D-tryptophane, D-Histidine and the D-proline(Pro);
Described side chain protected L-amino acid is O-protection-L-Serine, O-protection-L-Threonine, S-protection-L-halfcystine, N
ε-protection-L-Methionin, N
ω-protection-L-arginine, O-protection-L-tyrosine and N
ImIn-protection-L-the Histidine any one;
Described side chain protected D-amino acid is O-protection-D-Serine, O-protection-D-Threonine, S-protection-D-halfcystine, N
ε-protection-D-Methionin, N
ω-protection-D-arginine, O-protection-D-tyrosine and N
ImIn-protection-D-the Histidine any one.
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| CN101701239A (en) * | 2009-11-05 | 2010-05-05 | 南京工业大学 | Method for preparing glutathione by using hydantoinase method |
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