CN106279354A - A kind of synthetic method of amino acid neural dipeptides - Google Patents
A kind of synthetic method of amino acid neural dipeptides Download PDFInfo
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- CN106279354A CN106279354A CN201610880755.4A CN201610880755A CN106279354A CN 106279354 A CN106279354 A CN 106279354A CN 201610880755 A CN201610880755 A CN 201610880755A CN 106279354 A CN106279354 A CN 106279354A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06139—Dipeptides with the first amino acid being heterocyclic
- C07K5/06156—Dipeptides with the first amino acid being heterocyclic and Trp-amino acid; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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Abstract
nullThe invention discloses the synthetic method of a kind of amino acid neural dipeptides，Including: the synthesis of tertbutyloxycarbonyl phenylalanine arginine formicester (BOC Phe Arg OMe)、The synthesis of tertiary butyloxycarbonyl tryptophan glycine second fat (BOC Trp Gly OEt)、Tertiary butyloxycarbonyl tryptophan glycine second fat (BOC Trp Gly OEt) deprotection、BOC Phe Arg Trp Gly OEt synthesizes、Peptide prod is isolated and purified，The present invention is enzymatic clarification amino acid neural dipeptides in non-aqueous media，Organic solvent dimethylformamide (DMF) is selected during two peptide symthesis，And use in non-aqueous organic solvent, add triethylamine cosolvent and suitably increase water content，Increase the dissolubility of hydrophilic amino acid，Improve the availability of hydrophilic amino acid，Enzymatic clarification reaction condition is gentle，Stereospecificity is strong，Without Side chain protective group，Do not produce chiral molecule.
Description
Technical field
The invention belongs to peptide symthesis technical field, particularly relate to the synthetic method of a kind of amino acid neural dipeptides.
Background technology
The present Research of enzyme catalysis peptide symthesis: biologically active peptide is the material that a class is important, has each in vital movement
The biological function of kind of various kinds, but they in vivo content few, extract difficulty, synthesize them the most by artificial means, there is weight
The theory significance wanted and practical significance.The such as cause of hypertension, gastrointestinal disease, diabetes etc. is all relevant with bioactive peptide to treatment.
At present, abroad, especially U.S., day, De Deng developed country, the research of medical peptide is the most active.Biotechnology is in peptide method of production
Application created a collection for the treatment of disease true effective new drug in a new way.China's research in this field has
Development at full speed.Complex functionality small peptide, existing many reports, the synthesis of some functional oligopeptides has reached industrialized scale.Enzyme process
The peptides of synthesis primarily now concentrates on the polypeptide hormone being of practical significance and has nutritious peptides, and in these areas,
There is reported in literature both at home and abroad, the most successfully have the life utilizing thermolysin or papain to carry out Radix Asparagi saccharin
Producing, it is a kind of new food additive low in calories, particularly useful to diabetes patient.With papain and chymotrypsin protein
Enzymatic synthesis Leu and Met-enkelphalin, with carboxypeptidase-Y and chymotrypsin protein enzymatic synthesis Met-enkelphalin, with thermolysin,
The synthesis caerulin such as papain, subtilisin, pepsin etc..Remaining water field et al. is with Alcalse in ethanol
Synthesize containing the amino acid whose dipeptides of D type, theory and actual application has had important meaning.Biologically active peptide abroad
Study the most active, and create a collection of new drug in the application aspect of peptide development, bring great economic benefit.Japan Grnrtch
The Cervilaxin that company develops is used for treating wound and dwarfism, and the one that Telios drugmaker of the U.S. produces treats wound
Little peptide.Wound healing peptides reaches 1,000,000,000 dollars in U.S.'s annual sales amount.Application peptide medicament captures acquired immune deficiency syndrome (AIDS) HIV-1 to be become
New trial, the synthesis of biologically active peptide is also a field the most active, after chemical method has synthesized insulin, useful enzyme
Promote semi-synthesis method and be prepared for insulin analog.Additionally, in the synthesis of enkelphalin, atrial natriuretic peptide and the like, and Radix Ginseng is many
The development aspect of peptide yields good result.
At present peptide symthesis mainly has 3 kinds of methods: chemical method, recombinant DNA technology, enzyme process.In general, chemical method is in work
Still make with the most use in industry；Recombinant DNA technology is owing to needing long-term, an expensive research and development stage, and is sending out
There is low the extraction with product of expression efficiency and reclaim the problems such as difficult in the ferment stage, its application is very limited；Although enzyme process is started to walk
Later, but over nearly 20 years, many scientists have made considerable work, and because it is compared with chemical method, have side reaction
Less, reaction condition is gentle, regioselectivity is high and side chain needs less protection or need not the advantages such as protection, develops the most fast
Speed.Every kind of method has its merits and demerits, a kind of method of single employing always to have the shortcoming being difficult to overcome.Synthesis when concrete
Which kind of method synthesis is used mainly to see the length required by target product and purity.Chemo-enzymatic peptide synthesis completely is mainly used in little
The synthesis of peptide fragment (< 10) or the condensation of fragments of peptides, DNA recombinant technique is particularly suited for the conjunction containing hundreds of amino acid whose big peptides
Become, and for technically the most ripe chemical method synthesis, although solid phase method can be used to synthesis containing nearly 100 amino acid whose
Peptide, but it has most the application of Practical significance to be also used to synthesize the peptide fragment of moderate-length (10-100).Laboratory scale now
Upper synthetic peptide mainly be exactly chemical method, although it has two kinds of different methods of solid phase liquid phase, but both approaches is being changed
It is closely similar in process.
Thyroliberin (andrenocorticotropic hormone) i.e. ACTH or Corticotropin is
One contains the three amino acid whose direct-connected polypeptide of nineteen, and molecular weight is about 4700, sheep, pig, cattle ACTH the most purified, they are except the
Outside 25-33 aminoacid, remainder is the most identical, and the primary structure of ACTH is as follows: HN-silk-cheese-silk-egg-paddy-group-phenylpropyl alcohol-
Essence-Se-Gan-rely-dried meat-figured silk fabrics-Gan-Lai-rely-essence-essence-dried meat-figured silk fabrics-rely-figured silk fabrics-cheese-dried meat-Radix Asparagi-sweet-paddy-propyl-paddy-Radix Asparagi-silk-
Acrylate-paddy amine-propyl-phenylpropyl alcohol-dried meat-bright-paddy-phenylpropyl alcohol-COOH, interim sequence 1-24 amino acid residue has biological activity, and 4-10 is
Biological active center, is information position, and the combination to receptor provides key element again, and the 9th Trp of the 8th Arg is for starting receptor
One of active center, after the indole ring of Trp is modified (methylating or NPSization) or replaced by Phe, remain to and adrenal cortex
The adipose membrane of cell combines, but can not excite the release of c-AMP enzyme or 17-hydroxy-11-dehydrocorticosterone.9th Trp, the 6th His, the 5th Glu pair
The generation of corticosterone plays a crucial role.25-33 has species specificity and immunologic opsonin.Thyroliberin is by hypophysis
Anterior secrets the control by hypothalamus corticotropin-releasing factor (CRF).In turn, the target organ of ACTH is divided
The glucocorticoid steroid hormone secreted also has feedback effect to hypophysis and hypothalamus.First ACTH finds in hypophysis, afterwards at hypothalamus,
, with the presence of the immunoreactive bioactive peptide of ACHT, even also there is the existence of ACTH in brain district in gastrointestinal.Brain, hypothalamus, hypophysis
The ACTH of secretion performs different functions, and the Regulation Mechanism of the possible sense of participation behavior of the ACTH in brain, brain neuron is secreted
ACTH class bioactive peptide, be considered as the effector into neurotransmitter, affect the effect of pairing effect cell, they transmit signal
Apart from upper much shorter, institute's discharge stream much weaker.The ACTH of pituitary mainly acts on adrenal cortex, and it can promote adrenal gland
Cortex secreting hormone, and promote that the cholesterol of internal storage changes into Song bird in adrenal cortex.Stress mistake
Cheng Zhong, as calcination or damage time, the secretory volume of ACTH all dramatically increases, and this is shown to be whole body general mobilization, transfer neural with interior point
Secrete the positive movable of two aspects, ACTH secretory volume in brain with hypophysis all with stress degree be parallel relation, this also illustrates,
Neural activity is all related by ACTH race bioactive peptide with endocrine effect.
The thyroliberin (ACTH) used clinically at present, is 39 peptides extracted from the antepituitary of pig,
Or analog 18 peptide of chemosynthesis, 24 peptides, 28 peptides etc..Medical practice is applied ACTH diagnose adrenocortical
Physiological situation, due to the success of chemosynthesis ACTH, diagnosis and treatment rheumatic as hypercortisolism clinically is closed
Joint inflammation, dermatitis, ophthalmia and agaist allergic symptoms, also treat the illness such as gout, asthma and dermatodynia with it.It is lived research report
Property position heptapeptide (Met-Glu-His-Phe-Arg-Trp-Gly) is relevant with the behavior of people, can make the observation of compos mentis person
Power is improved, and the attention of people can be made to concentrate, can improve the emotion of people, make the faculty of understanding of amentia improve, moreover it is possible to make old people's
Hypermnesia, it can also the emotion of anxiety reduction.
Enzymatic clarification amino acid neural tetrapeptide Phe-Arg-Trp-Gly (FRWG) in non-aqueous media, from the point of view of methodology,
The enzymatic clarification of oligopeptide, relatively chemical method have obvious superiority, and are to rush to non-aqueous Enzyme catalyzed synthesis peptide bond technological difficulties
Hit, i.e. the utilization of hydrophilic amino acid.The synthesis of chiral drug is difficult point and the focus of current medical study on the synthesis, is also enzymatic
Synthesis is better than chemosynthesis and specifically embodies.It practice, target product is the chiral drug with significant application value, and make to have
The method synthesizing peptide bond in machine medium is more commonly changed and validation.Enzymatic clarification amino acid neural tetrapeptide Phe-in non-aqueous media
Arg-Trp-Gly (FRWG) is thyroliberin (ACTH) active site tetrapeptide, has significantly tune to nervous system
Joint effect, thus the peptide medicament new to exploitation, have extraordinary application prospect.
Summary of the invention
It is an object of the invention to provide that a kind of reaction condition is gentle, stereospecificity is strong, without Side chain protective group, a few nothing
Side reaction, than common chemical synthesis environmental protection, do not produce chiral molecule, need not split further, yield high and non-aqueous Jie
The method of enzymatic clarification amino acid neural dipeptides Phe-Arg-Trp-Gly in matter.
The present invention is achieved in that the synthetic method of a kind of amino acid neural dipeptides, the conjunction of this amino acid neural dipeptides
One-tenth method comprises the following steps:
The synthesis of tertbutyloxycarbonyl phenylalanine arginine formicester (BOC-Phe-Arg-OMe): tertbutyloxycarbonyl phenylpropyl alcohol ammonia
It is 0.53g (2mmol) that acid (BOC-Phe) adds quality, and it is 1.04g (4mmol) that arginine formicester (Arg-OMe) adds quality,
Organic solvent dimethylformamide (DMF) 20ml, 1ml triethylamine, a certain amount of tris-HCI buffer
(Tris-HCl buffer), adds chymase (Chymotyrpsin) 2g, and 40 DEG C of water-baths are vibrated 3 days, take 50 every 8h
μ l adds 500 μ l glacial acetic acids and terminates reaction, with the generation of Mass Spectrometer Method product；
The synthesis of tertiary butyloxycarbonyl tryptophan glycine second fat (BOC-Trp-Gly-OEt): BOC-Trp adds quality
It is 0.62g (4mmol) that 0.16g (2ml), Gly-OEt add quality, organic solvent dimethylformamide (DMF) 20ml, 1ml tri-
Ethamine, a certain amount of tris-HCI buffer (Tris-HClbuffer), add chymase
(Chymotyrpsin) 2g, 40 DEG C of water-baths are vibrated 3 days, take 50 μ l every 8h and add 500 μ l glacial acetic acids termination reactions, examine with mass spectrum
Survey the generation of product；
Tertbutyloxycarbonyl phenylalanine arginine formicester (BOC-Phe-Arg-OMe) and tertiary butyloxycarbonyl tryptophan glycine
The process of second fat (BOC-Trp-Gly-OEt): will be containing tertbutyloxycarbonyl phenylalanine arginine formicester (BOC-Phe-Arg-
OMe) and the organic solvent dimethylformamide (DMF) of tertiary butyloxycarbonyl tryptophan glycine second fat (BOC-Trp-Gly-OEt),
Decompression is distilled off organic solvent dimethylformamide (DMF) respectively, then with acetic acid ethyl dissolution, ethyl acetate uses cold lemon respectively
Lemon pickling three times, cold sodium bicarbonate is washed twice, then washes twice with cold water, decompression distillation, except ethyl acetate, obtains slightly yellow nothing fixed
Type thing；
Tertiary butyloxycarbonyl tryptophan glycine second fat (BOC-Trp-Gly-OEt) deprotection: tertiary butyloxycarbonyl tryptophan is sweet
Propylhomoserin second fat (BOC-Trp-Gly-OEt) product TFA (CF3COOH): methyl phenyl ethers anisole (9:1) just dissolves, 0 DEG C of vibration 2-4 is little
Addition ether time after: petroleum ether (1:3), obtains precipitation, centrifugal, adds by ether and petroleum ether mixing according to 1:3 composition of proportions
Close liquid and wash precipitation, obtain tryptophan glycine second fat (Trp-Gly-OEt)；
Peptide prod is isolated and purified: post material uses Sephadex G-10, and chromatographic column is 16mm × 1000mm, and eluent uses
Distilled water, detection wavelength is 220nm, and flow velocity is 1.0ml/min, takes 100g SephadexG-10 dry post material, adds 500ml distillation
Water soaking overnight, fills post after decompression bubble removing, and eluent balances overnight, and liquid 0.5ml loading to be separated chromatographs, and collects product
Peak, lyophilizing obtains product.
Further, the synthetic method of this amino acid neural dipeptides uses high-efficient liquid phase technique (HPLC) detection peptide symthesis purity,
The method of quantitative analysis peptide prod, for the analysis detection of synthetic peptide, uses high performance liquid chromatograph, and model is C18,250 ×
The Diamosil chromatographic column of 4.6mm, 220nm DAD UV-detector, column temperature 25 DEG C, solvent orange 2 A uses the second of 0.1% trifluoroacetic acid
Nitrile, solvent B uses the water containing 0.1% trifluoroacetic acid, and flow velocity is 1.0ml/min, and concrete gradient is:
Further, the synthetic method of this amino acid neural dipeptides uses mass spectrum to determine the method qualification peptide prod of molecular weight,
Mass spectrum (MS) condition is as follows: ionization mode: API ES；Polarity: positive；Voltage: 4000V；Nebulizer pressure: 35psig；Dry
Dry gas stream: 10L/min；Temperature: 350 DEG C；Sweep limits: 100-800a.m.u. (atomic mass units).
Further, the peptide prod of the synthetic method synthesis of this amino acid neural dipeptides coagulates with Sephedex G-10 molecular sieve
Plastic column chromatography separates, and collects product elution liquid peak, and after rotary evaporation concentrates, secondary repeats loading, after the eluent obtained concentrates,
Lyophilizing, utilizes the gross mass of container and product to deduct the difference assay of the quality of container own, calculates the weight of gained peptide prod, then
Utilize high-efficient liquid phase technique to detect, be multiplied by weight according to peptide prod purity, obtain the weight of pure products, this weight and theoretical yield
Ratio is exactly ultimate yield.
Further, chymase catalyzes and synthesizes dipeptides tertiary butyloxycarbonyl in organic solvent dimethylformamide (DMF)
During base phenylalanine arginine formicester (BOC-Phe-Arg-OMe), chymase and organic solvent dimethylformamide
(DMF) the synthetic system water content constituted 1.5%, reaction temperature 40 DEG C, pH be 7.5 time, tertbutyloxycarbonyl phenylalanine
Arginine formicester (BOC-Phe-Arg-OMe) productivity is the highest；
Chymase catalyzes and synthesizes dipeptides tertiary butyloxycarbonyl tryptophan in organic solvent dimethylformamide (DMF)
During glycine second fat (BOC-Trp-Gly-OEt), the conjunction that chymase and organic solvent dimethylformamide (DMF) are constituted
Architectonical water content 1.5%, reaction temperature 40 DEG C, pH be 8.0 time, tertiary butyloxycarbonyl tryptophan glycine second fat (BOC-
Trp-Gly-OEt) productivity is the highest.
Further, two peptide prod tertbutyloxycarbonyl phenylalanine essence ammonia of the synthetic method synthesis of this amino acid neural dipeptides
Through G-10 column chromatography after purification, molecular weight is 435.4, and quasi-molecular ions is 436.4 (M+1) in acid formicester (BOC-Phe-Arg-OMe)；
Synthesis two peptide prods tryptophan glycine second fat (Trp-Gly-OEt) through G-10 column chromatography after purification, molecule
Amount is 289.3, and quasi-molecular ions is 290.3 (M+1).
The synthetic method of the amino acid neural dipeptides that the present invention provides, including: tertbutyloxycarbonyl phenylalanine arginine first
The synthesis of fat (BOC-Phe-Arg-OMe), the synthesis of tertiary butyloxycarbonyl tryptophan glycine second fat (BOC-Trp-Gly-OEt),
Tertiary butyloxycarbonyl tryptophan glycine second fat (BOC-Trp-Gly-OEt) deprotection, BOC-Phe-Arg-Trp-Gly-OEt close
Become, peptide prod isolated and purified, the present invention is enzymatic clarification amino acid neural dipeptides in non-aqueous media, selects to have during two peptide symthesis
Machine solvent dimethylformamide (DMF), and use addition triethylamine cosolvent and suitably increase water in non-aqueous organic solvent to contain
Amount, increases the dissolubility of hydrophilic amino acid, improves the utilization rate of hydrophilic amino acid, and the present invention selects when two peptide symthesis to be had
Machine solvent dimethylformamide (DMF), and use addition triethylamine cosolvent and suitably increase water in non-aqueous organic solvent to contain
Amount, increases the dissolubility of hydrophilic amino acid, solves the Utilizing question of hydrophilic amino acid.Improve the utilization of hydrophilic amino acid
Degree, enzymatic clarification reaction condition is gentle, and stereospecificity is strong, without Side chain protective group, several without side reaction, than conventionally used change
Learn synthetic method environmental protection, do not produce chiral molecule, need not split further, improve yield, and optimize reaction condition.
Accompanying drawing explanation
Fig. 1 is the flowchart of the synthetic method of the amino acid neural dipeptides that the embodiment of the present invention provides；
Fig. 2 is that the water content that the embodiment of the present invention provides affects schematic diagram to dipeptides synthetic yield；
Fig. 3 is that the temperature that the embodiment of the present invention provides affects schematic diagram to dipeptides synthetic yield；
Fig. 4 is that the pH that the embodiment of the present invention provides affects schematic diagram to dipeptides synthetic yield；
Fig. 5 is the two peptide prod tertbutyloxycarbonyl phenylalanine arginine formicester (BOC-Phe-that the embodiment of the present invention provides
Arg-OMe) through G-10 column chromatography after purification HPLC figure；
Fig. 6 is two peptide prod tertbutyloxycarbonyl phenylalanine arginine formicesters of the synthesis that the embodiment of the present invention provides
(BOC-Phe-Arg-OMe) through G-10 column chromatography mass spectrum after purification；
Fig. 7 is two peptide prod tryptophan glycine second fat (Trp-Gly-OEt) warps of the synthesis that the embodiment of the present invention provides
Cross G-10 column chromatography HPLC figure after purification；
Fig. 8 is two peptide prods tryptophan glycine second fat (Trp-Gly-OEt) for synthesis that the embodiment of the present invention provides
Through G-10 column chromatography mass spectrum after purification.
Detailed description of the invention
In order to make the purpose of the present invention, technical scheme and advantage clearer, below in conjunction with embodiment, to the present invention
It is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not used to
Limit the present invention.
As it is shown in figure 1, the present invention is achieved in that the synthetic method of a kind of amino acid neural dipeptides, this aminoacid god
Comprise the following steps through the synthetic method of dipeptides:
Step S101, the synthesis of tertbutyloxycarbonyl phenylalanine arginine formicester (BOC-Phe-Arg-OMe): tertiary butyloxycarbonyl
It is 0.53g (2mmol) that base phenylalanine (BOC-Phe) adds quality, and it is 1.04g that arginine formicester (Arg-OMe) adds quality
(4mmol), organic solvent dimethylformamide (DMF) 20ml, 1ml triethylamine, a certain amount of trishydroxymethylaminomethane-hydrochloric acid
Buffer (Tris-HCl buffer), adds chymase (Chymotyrpsin) 2g, and 40 DEG C of water-baths are vibrated 3 days, every
8h takes 50 μ l and adds 500 μ l glacial acetic acids termination reactions, with the generation of Mass Spectrometer Method product；
Step S102, the synthesis of tertiary butyloxycarbonyl tryptophan glycine second fat (BOC-Trp-Gly-OEt): BOC-Trp adds
Enter quality be 0.16g (2ml), Gly-OEt add quality be 0.62g (4mmol), organic solvent dimethylformamide (DMF)
20ml, 1ml triethylamine, a certain amount of tris-HCI buffer (Tris-HCl buffer), add pancreas and coagulate
Galactase (Chymotyrpsin) 2g, 40 DEG C of water-baths are vibrated 3 days, take 50 μ l every 8h and add 500 μ l glacial acetic acids termination reactions,
Generation with Mass Spectrometer Method product；
Step S103, tertbutyloxycarbonyl phenylalanine arginine formicester (BOC-Phe-Arg-OMe) and tertiary butyloxycarbonyl primary colours
The process of propylhomoserin glycine second fat (BOC-Trp-Gly-OEt): will be containing tertbutyloxycarbonyl phenylalanine arginine formicester (BOC-
And the organic solvent dimethyl formyl of tertiary butyloxycarbonyl tryptophan glycine second fat (BOC-Trp-Gly-OEt) Phe-Arg-OMe)
Amine (DMF), decompression is distilled off organic solvent dimethylformamide (DMF) respectively, then with acetic acid ethyl dissolution, ethyl acetate is divided
Not with cold Fructus Citri Limoniae pickling three times, cold sodium bicarbonate is washed twice, then washes twice with cold water, decompression distillation, except ethyl acetate, obtains micro-
Yellow amorphous thing；
Step S104, tertiary butyloxycarbonyl tryptophan glycine second fat (BOC-Trp-Gly-OEt) deprotection: tertbutyloxycarbonyl
Tryptophan glycine second fat (BOC-Trp-Gly-OEt) product TFA (CF3COOH): methyl phenyl ethers anisole (9:1) just dissolves, 0 DEG C is shaken
Add ether after swinging 2-4 hour: petroleum ether (1:3), obtain precipitation, centrifugal, add by ether with petroleum ether according to 1:3 ratio structure
Precipitation washed by the mixed liquor become, and obtains tryptophan glycine second fat (Trp-Gly-OEt)；
Step S105, peptide prod is isolated and purified: post material uses Sephadex G-10, and chromatographic column is 16mm × 1000mm,
Eluent uses distilled water, and detection wavelength is 220nm, and flow velocity is 1.0ml/min, takes 100g Sephadex G-10 dry post material,
Adding 500ml distilled water immersion overnight, fill post after decompression bubble removing, eluent balances overnight, liquid 0.5ml loading layer to be separated
Analysis, collects product peak, and lyophilizing obtains product.
In embodiments of the present invention, the synthetic method of this amino acid neural dipeptides uses high-efficient liquid phase technique (HPLC) detection peptide
Synthesis purity, the method for quantitative analysis peptide prod, for the analysis detection of synthetic peptide, uses high performance liquid chromatograph, and model is
The Diamosil chromatographic column of C18,250 × 4.6mm, 220nm DAD UV-detector, column temperature 25 DEG C, solvent orange 2 A uses 0.1% 3
The acetonitrile of Fluoroethanoic acid, solvent B uses the water containing 0.1% trifluoroacetic acid, and flow velocity is 1.0ml/min, and concrete gradient is:
In embodiments of the present invention, the synthetic method of this amino acid neural dipeptides uses mass spectrum to determine the method mirror of molecular weight
Determining peptide prod, mass spectrum (MS) condition is as follows: ionization mode: API ES；Polarity: positive；Voltage: 4000V；Nebulizer pressure:
35psig；Dry air-flow: 10L/min；Temperature: 350 DEG C；Sweep limits: 100-800a.m.u. (atomic mass units).
In embodiments of the present invention, the peptide prod Sephedex G-of the synthetic method synthesis of this amino acid neural dipeptides
10 molecular sieve gel column chromatography for separation, collect product elution liquid peak, and after rotary evaporation concentrates, secondary repeats loading, the eluting obtained
After liquid concentrates, lyophilizing, utilize the gross mass of container and product to deduct the difference assay of the quality of container own, calculate gained peptide prod
Weight, recycling high-efficient liquid phase technique detection, be multiplied by weight according to peptide prod purity, obtain the weight of pure products, this weight with
The ratio of theoretical yield is exactly ultimate yield.
In embodiments of the present invention, chymase catalyzes and synthesizes two in organic solvent dimethylformamide (DMF)
During peptide tertbutyloxycarbonyl phenylalanine arginine formicester (BOC-Phe-Arg-OMe), chymase and organic solvent diformazan
The synthetic system water content that base Methanamide (DMF) is constituted 1.5%, reaction temperature 40 DEG C, pH be 7.5 time, tertbutyloxycarbonyl
Phenylalanine arginine formicester (BOC-Phe-Arg-OMe) productivity is the highest；
Chymase catalyzes and synthesizes dipeptides tertiary butyloxycarbonyl tryptophan in organic solvent dimethylformamide (DMF)
During glycine second fat (BOC-Trp-Gly-OEt), the conjunction that chymase and organic solvent dimethylformamide (DMF) are constituted
Architectonical water content 1.5%, reaction temperature 40 DEG C, pH be 8.0 time, tertiary butyloxycarbonyl tryptophan glycine second fat (BOC-
Trp-Gly-OEt) productivity is the highest.
In embodiments of the present invention, two peptide prod tertbutyloxycarbonyl benzene of the synthetic method synthesis of this amino acid neural dipeptides
Through G-10 column chromatography after purification, molecular weight is 435.4 to alanine arginine formicester (BOC-Phe-Arg-OMe), and quasi-molecular ions is
436.4(M+1)；
Synthesis two peptide prods tryptophan glycine second fat (Trp-Gly-OEt) through G-10 column chromatography after purification, molecule
Amount is 289.3, and quasi-molecular ions is 290.3 (M+1).
Below in conjunction with the accompanying drawings and the application principle of the present invention is further described by specific embodiment.
3 experimental sections
3.1 experiment material
3.1.1 aminoacid and derivant thereof
BOC-Phe；Arg-OMe.2HCl；BOC-Trp；Gly-OEt.HCl. (it is purchased from gill biochemical)
3.1.2 enzyme
Papain (Papain, Merch Germany,
Chymase (Chymotrypsin), Serva USA,
Trypsin trypsin), purchased from Beijing Ding Guo biotechnology Co., Ltd (Geview subpackage)
Alkaline protease Alcalase, purchased from Novo-Nordisk (Denmark)
3.1.3 reagent
Oxolane, dehydrated alcohol, absolute methanol, glacial acetic acid, concentrated hydrochloric acid, (from Shanghai, gill biochemistry is limited for trifluoroacetic acid
Responsible company), acetonitrile (chromatographic grade) (DIKMA, USA), sodium hydroxide, disodium hydrogen phosphate, sodium dihydrogen phosphate, dimethylformamide
(DMF), dilute hydrochloric acid, NaCl, sodium carbonate, sodium bicarbonate, trishydroxymethylaminomethane (Tris), ethyl acetate, isopropanol, Fructus Citri Limoniae
Acid, methyl phenyl ethers anisole, ether, petroleum ether, methanol, isobutanol, the tert-butyl alcohol etc. is analytical pure.
The most isolated and purified filler
Chromatographic stuffing SephadexG-10, Pharmacia
3.2 instrument
Power DryLL3000 freezer dryer (Heto company),
J2-21M High speed refrigerated centrifuge (Beckman, the U.S.),
JJ-1 type timing motor stirrer (Jiangsu Zhong great instrument plant),
PH S-2C type precision acidity meter (Shanghai Lei Ci instrument plant),
ESJ180-4 electronic balance (Longteng Electronic Weighing Instrument Co., Ltd., Shenyang),
ZFQ85A type Rotary Evaporators (Shanghai Medical Apparatus and Instruments Factory),
SHA-C type water-bath constant temperature oscillator (changzhou Guo Hua instrument plant),
Drying baker (Shanghai experimental apparatus head factory),
Chromatographic column (reversed-phase column Diamosil C18,250 × 4.6mm),
HD-2000 type UV-detector (Shanghai Jia Peng Science and Technology Ltd.),
Analytical type high pressure liquid chromatograph (the Waters U.S.),
LM17 type monitor (Yong Qing oscillograph factory),
P-1 type crossing current pump (Pharmacia Sweden),
HL-2 type crossing current pump (Industrial Co., Ltd. of upper Nereid section),
Electrospray mass spectrometer (Applied biosystems)
3.3 experimental technique
3.3.1 the enzyme' s catalysis of peptide
3.3.1.1 the enzyme' s catalysis of dipeptides
(1) synthesis of tertbutyloxycarbonyl phenylalanine arginine formicester (BOC-Phe-Arg-OMe)
It is 0.53g (2mmol) that tertbutyloxycarbonyl phenylalanine (BOC-Phe) adds quality, arginine formicester (Arg-OMe)
Addition quality is 1.04g (4mmol), organic solvent 20ml, 1ml triethylamine, and a certain amount of trishydroxymethylaminomethane-hydrochloric acid delays
Rushing liquid (Tris-HCl buffer), add enzyme 2g, 40 DEG C of water-baths are vibrated 3 days, take 50 μ l every 8h and add 500 μ l glacial acetic acids eventually
Only reaction, with the generation of Mass Spectrometer Method product.
(2) synthesis of tertiary butyloxycarbonyl tryptophan glycine second fat (BOC-Trp-Gly-OEt)
BOC-Trp add quality be 0.16g (2ml), Gly-OEt add quality be 0.62g (4mmol), organic solvent
20ml, 1ml triethylamine, a certain amount of tris-HCI buffer (Tris-HClbuffer), add enzyme 2g,
40 DEG C of water-baths are vibrated 3 days, take 50 μ l every 8h and add 500 μ l glacial acetic acids termination reactions, with the generation of Mass Spectrometer Method product.
3.3.1.2Phe-Arg-Trp-Gly the enzyme' s catalysis of tetrapeptide
(1) pretreatment of enzyme: take 2ml Alcalase and 15ml dehydrated alcohol is placed in centrifuge tube, vortex vibrates 5 minutes
Making it be sufficiently mixed, mixture 3000rpm is centrifuged 10 minutes, and enzyme is completely separable with solvent, the ethanol on upper strata of inclining, and this walks operation
In triplicate.
(2) dipeptides product treatment: will containing tertbutyloxycarbonyl phenylalanine arginine formicester (BOC-Phe-Arg-OMe) and
The organic solvent of tertiary butyloxycarbonyl tryptophan glycine second fat (BOC-Trp-Gly-OEt), decompression is distilled off organic molten respectively
Agent, product uses acetic acid ethyl dissolution again.Ethyl acetate is respectively with cold Fructus Citri Limoniae pickling three times, and cold sodium bicarbonate is washed twice, then uses
Cold water is washed twice, and decompression distillation, except ethyl acetate.Obtain slightly yellow amorphous article.
(3) tertiary butyloxycarbonyl tryptophan glycine second fat (BOC-Trp-Gly-OEt) deprotection: tertiary butyloxycarbonyl tryptophan
Glycine second fat (BOC-Trp-Gly-OEt) product TFA (CF3COOH): methyl phenyl ethers anisole (9:1) just dissolves, 0 DEG C of vibration 2-4
Addition ether after hour: petroleum ether (1:3), obtains precipitation, centrifugal.Add by ether and petroleum ether according to 1:3 composition of proportions
Precipitation washed by mixed liquor, obtains tryptophan glycine second fat (Trp-Gly-OEt).
(4) four peptide symthesis: take 2mmol tertbutyloxycarbonyl phenylalanine arginine formicester (BOC-Phe-Arg-OMe) and
Tryptophan glycine second fat (Trp-Gly-OEt) of 4mmol adds conical flask, adds organic solvent 20ml, enzyme 2ml, 1ml tri-second
Amine, a certain amount of Na2CO3-NaHCO3Buffer, 37 DEG C of water-baths are vibrated 24 hours, take 50 μ l every 4h and add 500 μ l glacial acetic acids
Terminate reaction, with the generation of Mass Spectrometer Method product.
(5) the same 3.3.1.2 of BOC-Phe-Arg-Trp-Gly-OEt tetrapeptide deprotection method (3) tertiary butyloxycarbonyl tryptophan
The method of glycine second fat (BOC-Trp-Gly-OEt) dipeptides deprotection, obtains Phe-Arg-Trp-Gly-OEt.
(6) Phe-Arg-Trp-Gly-OEt deprotection, takes step on 4.4g (5) Phe-Arg-Trp-Gly-OEt, adds
24ml methanol, 12.5mlNaOH (2mol/L), react one hour, adjust pH7 with HCl (1mol/L), pump methanol, dilute,
Filtering, ice bath cools down, and washing, to neutrality, obtains Phe-Arg-Trp-Gly Mass Spectrometer Method product.
3.3.2 peptide prod is isolated and purified
Post material: Sephadex G-10, chromatographic column: 16mm × 1000mm, eluent: distilled water, detect wavelength: 220nm,
Flow velocity: 1.0ml/min；
Take 100g Sephadex G-10 dry post material, add 500ml distilled water immersion overnight, after decompression bubble removing, fill post.Wash
De-liquid balances overnight.Liquid 0.5ml loading to be separated chromatographs.The separation being mainly used in synthesizing dipeptides, tetrapeptide and derivant thereof is pure
Change.Collecting product peak, lyophilizing obtains product.
3.3.3 the analysis of peptide prod
High-efficient liquid phase technique (HPLC) detection peptide symthesis purity:
The method of quantitative analysis peptide prod, the analysis for synthetic peptide detects,
High performance liquid chromatograph: Waters
Chromatographic column: C18,250 × 4.6mm, Diamosil
Detector: DAD UV-detector, 220nm
Column temperature: 25 DEG C
Elution requirement:
Solvent orange 2 A: the acetonitrile of 0.1% trifluoroacetic acid
The water of solvent B:0.1% trifluoroacetic acid
Flow velocity: 1.0ml/min.
3.3.4 the qualification of peptide prod
The main method using mass spectrum to determine molecular weight of qualification of product.Chemo-enzymatic peptide synthesis is because of its mild condition, and catalysis is specially
One property is strong, therefore does not substantially have the fracture of the internal covalent bond of aminoacid, and is substrate owing to using N end protected amino acid, substantially
Avoiding the generation of the by-product contrary with target product amino acid sequence, the method being therefore determined by molecular weight just can be reflected
Fixed output quota thing.Mass spectrum (MS) condition is as follows: ionization mode: API ES；Polarity: positive；Voltage: 4000V；Nebulizer pressure:
35psig；Dry air-flow: 10L/min；Temperature: 350 DEG C；Sweep limits: 100-800a.m.u. (atomic mass units).
3.3.5 calculation of yield
The peptide prod Sephedex G-10 molecular sieve gel column chromatography for separation of synthesis, collects product elution liquid peak, rotates
After evaporation and concentration, secondary repeats loading, after the eluent obtained concentrates, and lyophilizing, utilize difference assay (container and the gross mass of product
Deduct the quality of container itself) calculate the weight of products therefrom, recycling high-efficient liquid phase technique detection, it is multiplied by weight according to its purity
Amount, obtains the weight of pure products, and this weight is exactly ultimate yield with the ratio of theoretical yield.
4 results and discussion
4.1 enzymes and the selection of organic solvent
(1) synthesis of enzymatic dipeptides
Using different enzymes in different organic solvents, square method 3.3.1.1 of operating procedure, solution ph is all used
Tris-HCl buffer adjusts, and the organic solvent of water content and use is shown in Table 1, the results are shown in Table 1.
Table 1 enzyme catalysis two peptide symthesis
Note: F (BOC-Phe), R (Arg-OMe), F-R (BOC-Phe-ArgOMe)
W (BOC-Trp), G (Gly-OEt), W-G (tertiary butyloxycarbonyl tryptophan glycine second fat (BOC-Trp-Gly-
OEt))
Papain (papain), trypsin Trypsin)
Chymase (Chymotyrpsin), DMF (dimethylformamide)
Using the synthesis of four kinds of enzyme catalysis dipeptides, as can be seen from Table 1, generally, chemo-enzymatic peptide synthetic yield is at 20-
About 40%, but to catalyze and synthesize productivity in DMF higher for chymase, reaches more than 60%, illustrates that this system is suitable for
The synthesis of dipeptides.This is because the aromatic amino acids such as Phe and Trp are the hydrolysis substrates of chymase, with pancreas curdled milk egg
White enzyme active center matches, and the active center of chymase is beneficial to aromatic amino acid and enters, therefore synthetic yield meeting
The highest.And other enzyme is the most identical with the active sites of enzyme due to substrate, so productivity is relatively low.
(2) enzyme' s catalysis of BOC-Phe-Arg-Trp-Gly-OEt tetrapeptide
The synthesis of enzyme catalysis tetrapeptide, square method 3.3.1.2 (4), the enzymatic of BOC-Phe-Arg-Trp-Gly-OEt tetrapeptide closes
Become to the results are shown in Table 2.
Table 2 enzyme catalysis four peptide symthesis
To synthesize tetrapeptide productivity in the tert-butyl alcohol the highest for Alcalase as seen from Table 2.Alcalase is from bacillus subtilis
A kind of protease extracted, main component bacillus subtilis (Subtilisin) serine protease, is one of bacterialprotease, it
Good vigor can be kept in alcoholic solution.When four peptide symthesis, Alcalase is utilized to be catalyzed four peptide symthesis in the tert-butyl alcohol, by
Hydrophilic in t-butanol solvent, so two peptide substrates dissolubilities are higher than other organic solvent solubility, triethylamine also can promote the end
The dissolving of thing, additionally Alcalase is higher for the synthetic yield containing hydrophilic amino acid.Owing to solvent hydrophilic strengthens, Fructus Chaenomelis
Protease, trypsin and chymase vigor in the tert-butyl alcohol is lost a lot, and must be added in other solvent
A large amount of water make substrate dissolve very well, and otherwise productivity is the lowest.Meanwhile, two peptide substrates do not meet the active center of chymase
Structure, so productivity is not the highest.By experiment, have found the enzyme Alcalase that peptide symthesis is played a decisive role.
In chemo-enzymatic peptide synthesis, the selection of organic solvent is also critically important, this is because replace aqueous phase with organic solvent, flat
Weighing apparatus is carried out to peptide symthesis direction, after peptide generates, is diffused in organic solvent, and reaction just can be smoothed out.Enzyme is the most only at some
In organic solvent, ability is the most vibrant, and in other solvent, vigor is very little or none.For hydrophilic and the polarity situation of different solvents,
Synthesis dipeptides and tetrapeptide have selected different organic solvents respectively, and from the point of view of experimental result, chemo-enzymatic peptide is closed by these organic solvents
Becoming productivity to have a certain impact, wherein chymase productivity in dimethylformamide is higher, and Alcalase is at the tert-butyl alcohol
Middle productivity is higher.Selecting organic solvent to first have to consider its hydrophilic, hydrophilic organic solvent can dissolve hydrophilic amino
Acid, it is the most miscible with water that increase concentration of substrate just can improve productivity, DMF and the tert-butyl alcohol.Another Consideration is organic solvent
Boiling point, when product processes need decompression distillation or rotary evaporation, if boiling point is too high, product separate highly difficult.DMF and tertiary fourth
Alcohol boiling point is at about 100 DEG C.Additionally it is also considered that organic solvent toxicity, select the organic solvent that toxicity is less, DMF as far as possible
The least with the toxicity of the tert-butyl alcohol.
The impact on peptide symthesis of the 4.2 system water contents
Fig. 2 is that water content affects schematic diagram to dipeptides synthetic yield, from figure 2, it is seen that water content is when 1.5%, and dipeptides
Productivity reaches the highest.Water in Organic Solvents content is very important on the impact of enzymatic activity, and the water in usual system is with three kinds
Mode exists: the water being combined with enzyme, with the combination water of water-insoluble, free water in a solvent.Enzyme is catalyzed in organic solvent
Having an optimum moisture content, when water content is higher than the suitableeest water content, water can be competed the binding site of nucleopilic reagent, cause at the bottom of ester
Thing hydrolyzes, and reduces the yield of target product, the enzyme conformation excessively rigidity when water content is less than the suitableeest water content, simultaneously solvent
The highly polar necessary water having captured enzyme, causes the catalysis activity of enzyme to reduce and even inactivates, and only under being best suitable for water content, enzyme divides
The existing certain rigidity of son, has again certain flexibility, and peptide productivity could be the highest.The water content of system has with the hydrophobicity of solvent
Closing, solvent is the most hydrophilic, and system water content is the most, because solvent is hydrophilic, the ability of the necessary water of contention enzyme is the strongest, the most
Add a little water, the activity of guarantee enzyme.This be also water content in Alcalase-tert-butyl alcohol system higher than chymase-
One of reason of DMF content of water in system, the hydrophilic of the tert-butyl alcohol is greater than the hydrophilic of DMF.It addition, the water in reaction system contains
Amount not only affects the catalysis activity of enzyme, has an effect on the stereo selectivity of enzyme, this is because water changes the flexibility of enzyme molecule, mapping
Selectivity and the water content of body have dependence.
The impact of 4.3 reaction temperatures
Fig. 3 is that temperature affects schematic diagram to dipeptides synthetic yield, as can be seen from Figure 3, when reaction temperature is at 40 DEG C, and dipeptides
Productivity reaches the highest.Generally improve reaction temperature and can improve enzymatic response speed.Temperature is too low, and response speed is the slowest；
But the too high meeting of temperature causes enzyme denaturation, enzyme catalysis vigor is caused to be decreased obviously.Target peptide is closed by this study tour reaction temperature
Become the impact of yield.Thinking to be at enzyme molecule and be best suitable for temperature, vigor reaches the highest.Finding in an experiment, enzyme is having
Optimum temperature in machine solvent is higher than the optimum temperature of aqueous phase, this is because enzyme molecule electrostatic interaction in organic solvent
The electrostatic interaction being greater than in aqueous phase, molecular rigidity strengthens, flexible reduction, and its heat stability improves, and this is also that enzyme is organic
Solvent is catalyzed an advantage of peptide symthesis.
The impact of 4.4 systems pH
Fig. 4 is that pH affects schematic diagram to dipeptides synthetic yield, as seen from Figure 4, when pH is 7.5, and tertbutyloxycarbonyl
Phenylalanine arginine formicester (BOC-Phe-Arg-OMe) productivity reaches the highest, when pH is 8.0, and tertiary butyloxycarbonyl tryptophan
Glycine second fat (BOC-Trp-Gly-OEt) synthetic yield reaches the highest.Enzyme also has optimum pH scope in organic solvent, with water
Compare difference little.PH can affect the dissociated state of substrate molecule, also can affect the dissociated state of enzyme molecule, optimum pH with
The relation of having dissociated of the two, under optimum pH, the active center molecule of substrate molecule and enzyme is in the easiest bonding state, at this
Under the conditions of enzyme activity the highest, can well combine formation compound intermediate, beneficially peptide symthesis.It addition, pH in organic solvent
Affect dissociating of enzyme some groups of molecule, be that the conformation of enzyme changes so that it is optimum pH changes, also affect peptide symthesis
Productivity.
The analysis of 4.5 peptide prods and qualification
Fig. 5 is shown that two peptide prod tertbutyloxycarbonyl phenylalanine arginine formicester (BOC-Phe-Arg-of synthesis
OMe) through G-10 column chromatography after purification HPLC figure.
Fig. 6 is that two peptide prods tertbutyloxycarbonyl phenylalanine arginine formicester (BOC-Phe-Arg-OMe) of synthesis pass through
G-10 column chromatography mass spectrum after purification, molecular weight is 435.4, and in mass spectrum, quasi-molecular ions is 436.4 (M+1).
Fig. 7. the HPLC figure of tryptophan glycine second fat (Trp-Gly-OEt)
Fig. 7 be synthesis two peptide prods tryptophan glycine second fat (Trp-Gly-OEt) through G-10 column chromatography after purification
HPLC figure.
Fig. 8 be synthesis two peptide prods tryptophan glycine second fat (Trp-Gly-OEt) through G-10 column chromatography after purification
Mass spectrum, molecular weight is 289.3, and in mass spectrum, quasi-molecular ions is 290.3 (M+1).
5 conclusions
In the peptide symthesis with amino acid neural tetrapeptide Phe-Arg-Trp-Gly as target is studied, investigate peptide symthesis
Influential many factors, first have found optimal enzyme to be catalyzed the synthesis of peptide, secondly find that organic solvent is to peptide symthesis
Productivity also has an impact, and have found applicable organic solvent DMF and the tert-butyl alcohol.Change system water content, pH and reaction in an experiment
Temperature finds optimum reaction condition, constructs optimum response model, makes the productivity of dipeptides arrive separately at 61.25%, and 85.6%,
Tetrapeptide productivity also reaches 68.32%.
The problem that the invention solves the problems that is the Utilizing question of hydrophilic substrates, then first have to consider hydrophilic substrates
Dissolubility in organic solvent, this is necessary for selecting suitable organic solvent, selects DMF, secondly add three second during two peptide symthesis
Amine, its objective is to increase the dissolubility of substrate, contributes to the carrying out of reaction.
The synthetic method of the amino acid neural dipeptides that the embodiment of the present invention provides, including: tertbutyloxycarbonyl phenylalanine essence
The synthesis of propylhomoserin formicester (BOC-Phe-Arg-OMe), tertiary butyloxycarbonyl tryptophan glycine second fat (BOC-Trp-Gly-OEt)
Synthesis, tertiary butyloxycarbonyl tryptophan glycine second fat (BOC-Trp-Gly-OEt) deprotection, BOC-Phe-Arg-Trp-Gly-
OEt synthesis, peptide prod are isolated and purified, and the present invention is enzymatic clarification amino acid neural dipeptides in non-aqueous media, during two peptide symthesis
Select organic solvent dimethylformamide (DMF), and use addition triethylamine cosolvent and suitably increasing in non-aqueous organic solvent
Add water content, increase the dissolubility of hydrophilic amino acid, solve the Utilizing question of hydrophilic amino acid.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all essences in the present invention
Any amendment, equivalent and the improvement etc. made within god and principle, should be included within the scope of the present invention.
Claims (6)
1. the synthetic method of an amino acid neural dipeptides, it is characterised in that the synthetic method of this amino acid neural dipeptides includes
Following steps:
The synthesis of tertbutyloxycarbonyl phenylalanine arginine formicester: it is 0.53g that tertbutyloxycarbonyl phenylalanine adds quality, essence ammonia
It is 1.04g that acid formicester adds quality, organic solvent dimethylformamide 20ml, 1ml triethylamine, trishydroxymethylaminomethane-salt
Acid buffer, adds chymase 2g, and 40 DEG C of water-baths are vibrated 3 days, takes 50 μ l every 8h and adds 500 μ l glacial acetic acids terminations instead
Should, with the generation of Mass Spectrometer Method product；
It is that 0.16g, Gly-OEt addition quality is that the synthesis of tertiary butyloxycarbonyl tryptophan glycine second fat: BOC-Trp adds quality
0.62g, organic solvent dimethylformamide 20ml, 1ml triethylamine, tris-HCI buffer, add pancreas and coagulate
Galactase g, 40 DEG C of water-baths are vibrated 3 days, take 50 μ l every 8h and add 500 μ l glacial acetic acids termination reactions, use Mass Spectrometer Method product
Generation；
Tertbutyloxycarbonyl phenylalanine arginine formicester and the process of tertiary butyloxycarbonyl tryptophan glycine second fat: will be containing tertiary fourth
Oxygen carbonyl phenylalanine arginine formicester and the organic solvent dimethylformamide of tertiary butyloxycarbonyl tryptophan glycine second fat, point
Organic solvent dimethylformamide Jian Ya not be distilled off, then with acetic acid ethyl dissolution, ethyl acetate is respectively with cold Fructus Citri Limoniae pickling
Three times, cold sodium bicarbonate is washed twice, then washes twice with cold water, decompression distillation, except ethyl acetate, obtains slightly yellow amorphous article；
Tertiary butyloxycarbonyl tryptophan glycine second seborrheic alopecia is protected: tertiary butyloxycarbonyl tryptophan glycine second fat product TFA: benzene first
Ether=9:1 just dissolves, and 0 DEG C of vibration adds ether after 2-4 hour: petroleum ether=1:3, obtains precipitation, centrifugal, adds by ether
Wash precipitation with petroleum ether according to the mixed liquor of 1:3 composition of proportions, obtain tryptophan glycine second fat；
Peptide prod is isolated and purified: post material uses Sephadex G-10, and chromatographic column is 16mm × 1000mm, and eluent uses distillation
Water, detection wavelength is 220nm, and flow velocity is 1.0ml/min, takes 100g SephadexG-10 dry post material, adds 500ml and distills water logging
Bubble overnight, fills post after decompression bubble removing, and eluent balances overnight, and liquid 0.5ml loading to be separated chromatographs, and collects product peak, freezes
Do to obtain product.
2. the synthetic method of amino acid neural dipeptides as claimed in claim 1, it is characterised in that this amino acid neural dipeptides
Synthetic method uses high-efficient liquid phase technique detection peptide symthesis purity, and the method for quantitative analysis peptide prod is for the analysis inspection of synthetic peptide
Surveying, use high performance liquid chromatograph, model is the Diamosil chromatographic column of C18,250 × 4.6mm, 220nm DAD ultraviolet detection
Device, column temperature 25 DEG C, solvent orange 2 A uses the acetonitrile of 0.1% trifluoroacetic acid, and solvent B uses the water containing 0.1% trifluoroacetic acid, and flow velocity is
1.0ml/min。
3. the synthetic method of amino acid neural dipeptides as claimed in claim 1, it is characterised in that this amino acid neural dipeptides
Synthetic method uses mass spectrum to determine the method qualification peptide prod of molecular weight, and Mass Spectrometry Conditions is as follows: ionization mode: API ES；Pole
Property: positive；Voltage: 4000V；Nebulizer pressure: 35psig；Dry air-flow: 10L/min；Temperature: 350 DEG C；Sweep limits:
100-800a.m.u。
4. the synthetic method of amino acid neural dipeptides as claimed in claim 1, it is characterised in that this amino acid neural dipeptides
The peptide prod Sephedex G-10 molecular sieve gel column chromatography for separation of synthetic method synthesis, collects product elution liquid peak, rotates
After evaporation and concentration, secondary repeats loading, after the eluent obtained concentrates, and lyophilizing, utilize container to deduct container with the gross mass of product
The difference assay of quality own, calculates the weight of gained peptide prod, recycling high-efficient liquid phase technique detection, takes advantage of according to peptide prod purity
With weight, obtaining the weight of pure products, this weight is exactly ultimate yield with the ratio of theoretical yield.
5. the synthetic method of amino acid neural dipeptides as claimed in claim 1, it is characterised in that chymase is organic
When solvent dimethylformamide catalyzes and synthesizes dipeptides tertbutyloxycarbonyl phenylalanine arginine formicester, chymase with have
The synthetic system water content that machine solvent dimethylformamide is constituted 1.5%, reaction temperature 40 DEG C, pH be 7.5 time, tertiary fourth oxygen
Carbonyl phenylalanine arginine formicester productivity is the highest；
Chymase catalyzes and synthesizes dipeptides tertiary butyloxycarbonyl tryptophan glycine second in organic solvent dimethylformamide
During fat, the synthetic system water content that chymase and organic solvent dimethylformamide are constituted 1.5%, reaction temperature exists
40 DEG C, pH is when being 8.0, tertiary butyloxycarbonyl tryptophan glycine second fat productivity is the highest.
6. the synthetic method of amino acid neural dipeptides as claimed in claim 1, it is characterised in that this amino acid neural dipeptides
Synthetic method synthesis two peptide prod tertbutyloxycarbonyl phenylalanine arginine formicesters through G-10 column chromatography after purification, molecular weight
Being 435.4, quasi-molecular ions is 436.4；
Through G-10 column chromatography after purification, molecular weight is 289.3 to two peptide prod tryptophan glycine second fat of synthesis, and quasi-molecular ions is
290.3。
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| CN101570570A (en) * | 2009-06-03 | 2009-11-04 | 兰州大学 | Synthetic method for somatostatin |
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| CN101570570A (en) * | 2009-06-03 | 2009-11-04 | 兰州大学 | Synthetic method for somatostatin |
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