CN106478771A - A kind of synthetic method of amino acid neural tetrapeptide - Google Patents
A kind of synthetic method of amino acid neural tetrapeptide Download PDFInfo
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Abstract
The invention discloses a kind of synthetic method of amino acid neural tetrapeptide, including：The synthesis of tertbutyloxycarbonyl phenylalanine arginine formicester, the synthesis of tertiary butyloxycarbonyl tryptophan glycine second fat, the pretreatment of alkali protease, tertiary butyloxycarbonyl tryptophan glycine second fat deprotection, tertbutyloxycarbonyl phenylalanine arginine tryptophan glycine second fat synthesizes, tertbutyloxycarbonyl phenylalanine arginine tryptophan glycine second fat deprotection, phenylalanine arginine tryptophan glycine second fat deprotection, peptide prod is isolated and purified, present invention enzymatic clarification amino acid neural tetrapeptide Phe Arg Trp Gly in non-aqueous media, the organic solvent tert-butyl alcohol is selected during four peptide symthesis, and using addition triethylamine cosolvent in non-aqueous organic solvent and appropriate increase water content, to increase the solubility of hydrophilic amino acid, solve the Utilizing question of hydrophilic amino acid.
Description
Technical field
The invention belongs to peptide symthesis technology field, more particularly to a kind of synthetic method of amino acid neural tetrapeptide.
Background technology
The present Research of enzymatic peptide symthesis：Biologically active peptide is the important material of a class, has each in vital movement
The biological function of kind of various kinds, but they in vivo content few, extract difficult, therefore synthesize them by artificial means, with weight
The theory significance that wants and practical significance.The such as cause of hypertension, gastrointestinal disease, diabetes etc. is related all with active peptide to treatment.
At present, abroad, especially U.S., day, De Deng developed country, the research of medical peptide enliven very much.Biotechnology is in peptide method of production
Application generated a collection for the treatment of disease true effective new drug in a new way.Research of the China in this field has
Development at full speed.Complex functionality small peptide, existing many reports, some functional oligopeptides synthesis have reached industrialized scale.Enzyme process
The peptides of synthesis primarily now concentrate on the polypeptide hormone that is of practical significance and the nutritious peptides of tool, and in these areas,
There is reported in literature both at home and abroad, wherein more successfully have carries out the life of asparagus fern saccharin using thermolysin or papain
Produce, 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,
Synthesis caerulin such as papain, subtilopeptidase A, pepsin etc..Remaining water field et al. Alcalse is in ethanol
Synthesize the dipeptides containing D type amino acid, have important meaning in theory and actual application.Biologically active peptide abroad
Research is enlivened very much, and the application aspect in peptide development generates a collection of new drug, brings great economic benefit.Japanese Grnrtch
The relaxin that company develops is used for controlling treatment trauma and nanism, and one kind of Telios drugmaker of U.S. production controls treatment trauma
Small peptide.Wound healing peptides reach 1,000,000,000 dollars in U.S.'s annual sales amount.Application peptide medicament captures AIDS HIV-1 to be become
New trial, the synthesis of biologically active peptide is also a very active field, 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 ginseng is many
Yield good result in terms of the development of peptide.
Peptide symthesis mainly have 3 kinds of methods at present：Chemical method, recombinant DNA technology, enzyme process.In general, chemical method is in work
Still make in industry with the most use；Recombinant DNA technology is due to needing a long-term, expensive research and development stage, and is sending out
The ferment stage has that the low and product of expression efficiency is extracted and reclaims that 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 which is compared with chemical method, with side reaction
The advantage such as less, reaction condition is gentle, regioselectivity is high and side chain needs less protection or need not protect, develops quite fast
Speed.Every kind of method has its merits and demerits, and list is always difficult to overcome using a kind of method.Concrete when synthesis
Which kind of method synthesis length and purity required by target product are mainly seen using.Completely enzymatic peptide symthesis are mainly used in little
Peptide fragment (<10) synthesis or the condensation of fragments of peptides, DNA recombinant technique are particularly suited for the conjunction of the big peptide containing hundreds of amino acid
Become, and for technically ripe chemical method synthesis, although solid phase method can be used to synthesize containing nearly 100 amino acid
Peptide, but its application most of practical meaning is also used to synthesize the peptide fragment of moderate-length (10-100).Present laboratory scale
Upper synthetic peptide mainly be exactly chemical method, although it has two kinds of different methods of solid phase liquid phase, but both approaches are being changed
It is closely similar in process.
Corticotropin (andrenocorticotropic hormone) i.e. ACTH or Corticotropin is
One direct-connected polypeptide containing three nineteen amino acid, molecular weight are about 4700, sheep, pig, ox ACTH purified, they are except the
Outside 25-33 amino acid, remainder is all identical, and the primary structure of ACTH is as follows：HN- silk-junket-silk-egg-paddy-group-phenylpropyl alcohol-
Essence-Se-Gan-rely-dried meat-figured silk fabrics-Gan-Lai-bad-essence-essence-dried meat-figured silk fabrics-to rely the sweet-paddy -propyl- paddy-asparagus fern-silk-of-figured silk fabrics-junket-dried meat-asparagus fern-
Propyl- paddy amine -propyl- phenylpropyl alcohol-dried meat-bright-paddy-phenylpropyl alcohol-COOH, interim sequence 1-24 amino acid residue have biologically active, and 4-10 is
Biological active center, is both information position, and the combination to acceptor provides key element, and the 9th Trp of the 8th Arg is for starting acceptor
One of activated centre, (methylate or NPSization) after the indole ring of Trp is modified or replaced by Phe, remain to and adrenal cortex
The adipose membrane of cell is combined, but can not excite the release of c-AMP enzyme or cortin.9th Trp, the 6th His, the 5th Glu pair
The generation of cortisone plays a crucial role.25-33 has species specificity and immunologic opsonin.Corticotropin is by hypophysis
Anterior secrets the control by hypothalamus cortico-trophin-releasing factor (CRF) (CRF).In turn, the target organ of ACTH is divided
The glucocorticoid steroid hormone that secretes also has feedback effect to hypophysis and hypothalamus.ACTH is had found first in hypophysis, afterwards in hypothalamus,
Brain area is with the presence of the immunoreactive active peptide of ACHT, or even also has the presence of ACTH in stomach and intestine.Brain, hypothalamus, hypophysis
The ACTH of secretion executes different functions, and the Regulation Mechanism of the possible sense of participation behavior of the ACTH in brain, brain neuron are secreted
ACTH class active peptide, the effector being considered as neurotransmitter, affect pairing effect cell effect, 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 the cholesterol of internal storage to change into Song bird in adrenal cortex.Stress mistake
Cheng Zhong, such as calcination or when damaging, the secretory volume of ACTH is all dramatically increased, and this is shown to be whole body general mobilization, transfer neural with interior point
Secrete the positive activity of two aspects, secretory volume of the ACTH in brain and hypophysis all with stress degree be in parallel relation, this also illustrates,
ACTH race active peptide is all related with endocrine effect to nervous activity.
The corticotropin (ACTH) for clinically using at present, is 39 peptides extracted from the anterior pituitary of pig,
Or 18 peptide of analog of chemical synthesis, 24 peptides, 28 peptides etc..On medical practice, application ACTH is adrenocortical to diagnose
Physiological situation, due to the success of chemical synthesis ACTH, clinically the diagnosis as hypercortisolism and treatment rheumatic are closed
Section inflammation, dermatitis, ophthalmia and agaist allergic symptoms, also treat the illness such as gout, asthma and dermatalgia with it.There is its work of 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 improves, and concentrates can the notice of people, can improve the mood of people, improve the comprehension of amentia, moreover it is possible to make the elderly's
Hypermnesia, it can also anxiety reduction mood.
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 oligopeptides, has obvious superiority compared with chemical method, and is rushed 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 the difficult point of current medical study on the synthesis and focus, and enzymatic
Synthesis is embodied better than chemical synthesis.In fact, target product is the chiral drug with significant application value, and make to have
The method for 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 corticotropin (ACTH) active site tetrapeptide, nervous system is had and is significantly adjusted
Section is acted on, thus to developing new peptide medicament, with extraordinary application prospect.
Content of the invention
It is an object of the invention to provide a kind of reaction condition is gentle, stereocpecificity strong, without Side chain protective group, a few nothings
Side reaction, than common chemical synthesis environmental protection, do not produce chiral molecules, without splitting further, high income in non-aqueous Jie
The method of enzymatic clarification amino acid neural tetrapeptide Phe-Arg-Trp-Gly in matter.
The present invention is achieved in that a kind of synthetic method of amino acid neural tetrapeptide, the conjunction of the amino acid neural tetrapeptide
Method is become to comprise the following steps：
The synthesis of tertbutyloxycarbonyl phenylalanine arginine formicester (BOC-Phe-Arg-OMe)：Tertbutyloxycarbonyl phenylpropyl alcohol ammonia
It is 0.53g (2mmol) that sour (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 chymotrypsin (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 acid terminating reactions, with the generation of Mass Spectrometer Method product；
The synthesis of tertiary butyloxycarbonyl tryptophan glycine second fat (BOC-Trp-Gly-OEt)：BOC-Trp add quality be
It is 0.62g (4mmol), organic solvent dimethylformamide (DMF) 20ml, 1ml tri- that 0.16g (2ml), Gly-OEt add quality
Ethamine, a certain amount of tris-HCI buffer (Tris-HCl buffer), add chymotrypsin
(Chymotyrpsin) 2g, 40 DEG C of water-baths vibrate 3 days, take 50 μ l every 8h and add 500 μ l glacial acetic acid terminating reactions, are examined with mass spectrum
Survey the generation of product；
The pretreatment of alkali protease (Alcalase)：Take 2ml alkali protease (Alcalase) and 15ml absolute ethyl alcohol
It is placed in centrifuge tube, vortex vibration is sufficiently mixed which in 5 minutes, and mixture 3000rpm is centrifuged 10 minutes, alkali protease
(Alcalase) completely separable with solvent absolute ethyl alcohol, the ethanol on upper strata of inclining, this step operation is in triplicate；
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 tertiary butyloxycarbonyl tryptophan glycine second fat (BOC-Trp-Gly-OEt) organic solvent dimethylformamide (DMF),
Vacuum distillation removes organic solvent dimethylformamide (DMF) respectively, then is dissolved with ethyl acetate, and ethyl acetate uses cold lemon respectively
Lemon pickling three times, cold sodium acid carbonate is washed twice, then is washed twice with cold water, vacuum 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 dissolve, 0 DEG C of vibration 2-4 is little
When after add ether：Petroleum ether (1:3), must precipitate, centrifugation, add by ether and petroleum ether according to 1:The mixing of 3 ratios is constituted
Mixed liquor wash precipitation, obtain tryptophan glycine second fat (Trp-Gly-OEt)；
Tertbutyloxycarbonyl phenylalanine arginine tryptophan glycine second fat (BOC-Phe-Arg-Trp-Gly-OEt) is closed
Become：Take the sweet ammonia of tryptophan of tertbutyloxycarbonyl phenylalanine arginine formicester (BOC-Phe-Arg-OMe) of 2mmol and 4mmol
Sour second fat (Trp-Gly-OEt) adds conical flask, adds organic solvent tert-butyl alcohol 20ml, alkali protease (Alcalase) 2ml,
1ml triethylamine, a certain amount of Na2CO3-NaHCO3Buffer solution, 37 DEG C of water-baths vibrate 24 hours, take 50 μ l every 4h and add 500 μ l
Glacial acetic acid terminating reaction, with the generation of Mass Spectrometer Method product；
Tertbutyloxycarbonyl phenylalanine arginine tryptophan glycine second fat (BOC-Phe-Arg-Trp-Gly-OEt) remove-insurance
Shield：Tertbutyloxycarbonyl phenylalanine arginine tryptophan glycine second fat (BOC-Phe-Arg-Trp-Gly-OEt) product TFA
(CF3COOH):Methyl phenyl ethers anisole (9:1) just dissolve, after 0 DEG C of vibration 2-4 hour, add ether：Petroleum ether (1:3), must precipitate, from
The heart, adds by ether and petroleum ether according to 1:Precipitation washed by the mixed liquor that the mixing of 3 ratios is constituted, and obtains phenylalanine arginine color ammonia
Sour glycine second fat (Phe-Arg-Trp-Gly-OEt)；
Phenylalanine arginine tryptophan glycine second fat (Phe-Arg-Trp-Gly-OEt) deprotection：Take 4.4g phenylpropyl alcohol
Propylhomoserin arginine tryptophan glycine second fat (Phe-Arg-Trp-Gly-OEt), adds 24ml methyl alcohol, 12.5mlNaOH (2mol/
L), reacting one hour, pH being adjusted to 7 with HCl (1mol/L), pump methyl alcohol, be diluted with water, filter, ice bath is cooled down, in washing
Property, amino acid neural tetrapeptide (Phe-Arg-Trp-Gly) is obtained, uses Mass Spectrometer Method product；
Peptide prod is isolated and purified：Post material adopts Sephadex G-10, and chromatographic column is 16mm × 1000mm, and eluent is adopted
Distilled water, Detection wavelength are 220nm, and flow velocity is 1.0ml/min, take the dry post material of 100g Sephadex G-10, plus 500ml distillation
Water soaked overnight, fills post after decompression bubble removing, and eluent is balanced overnight, liquid 0.5ml loading chromatography to be separated, collects product
Peak, is lyophilized to obtain product.
Further, the synthetic method of the amino acid neural tetrapeptide detects peptide symthesis purity using high-efficient liquid phase technique (HPLC),
The method of quantitative analysis peptide prod is used for the analysis of synthetic peptide and detects, using high performance liquid chromatograph, model C18,250 ×
The Diamosil chromatographic column of 4.6mm, 220nm DAD UV-detector, 25 DEG C of column temperature, solvent orange 2 A is using the second of 0.1% trifluoroacetic acid
Nitrile, using the water containing 0.1% trifluoroacetic acid, flow velocity is 1.0ml/min to solvent B, and concrete gradient is：
Further, the synthetic method of the amino acid neural tetrapeptide determines the method identification peptide prod of molecular weight using mass spectrum,
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℃；Sweep limits：100-800a.m.u.(atomic mass units).
Further, the peptide prod of the synthetic method synthesis of the amino acid neural tetrapeptide is solidifying with Sephedex G-10 molecular sieve
Plastic column chromatography is separated, collection product elution liquid peak, secondary repetition loading after rotary evaporation concentration, after the eluent for obtaining is concentrated,
Lyophilized, the difference assay of container quality itself is deducted using the gross mass of container and product, calculates the weight of gained peptide prod, then
Detected using high-efficient liquid phase technique, weight is multiplied by according to peptide prod purity, the weight of pure products is obtained, this weight and theoretical yield
Ratio is exactly ultimate yield.
Further, chymotrypsin catalyzes and synthesizes dipeptides tertiary butyloxycarbonyl in organic solvent dimethylformamide (DMF)
During base phenylalanine arginine formicester (BOC-Phe-Arg-OMe), chymotrypsin and organic solvent dimethylformamide
(DMF) the synthetic system water content for constituting 1.5%, reaction temperature 40 DEG C, pH be 7.5 when, tertbutyloxycarbonyl phenylalanine
Arginine formicester (BOC-Phe-Arg-OMe) yield highest；
Chymotrypsin catalyzes and synthesizes dipeptides tertiary butyloxycarbonyl tryptophan in organic solvent dimethylformamide (DMF)
During glycine second fat (BOC-Trp-Gly-OEt), the conjunction that chymotrypsin is constituted with organic solvent dimethylformamide (DMF)
Architectonical water content 1.5%, reaction temperature 40 DEG C, pH be 8.0 when, tertiary butyloxycarbonyl tryptophan glycine second fat (BOC-
Trp-Gly-OEt) yield highest；
Alkali protease (Alcalase) catalyzes and synthesizes tetrapeptide tertbutyloxycarbonyl phenylalanine in the organic solvent tert-butyl alcohol
During arginine tryptophan glycine second fat (BOC-Phe-Arg-Trp-Gly-OEt), alkali protease (Alcalase) with organic
Solvent tertiary butanol constitute synthetic system water content 9%, reaction temperature 37 DEG C, pH be 9.0 when, tetrapeptide tertbutyloxycarbonyl benzene
Alanine arginine tryptophan glycine second fat (BOC-Phe-Arg-Trp-Gly-OEt) synthetic yield highest.
Further, the two peptide prod tertbutyloxycarbonyl phenylalanines essence ammonia of the synthetic method synthesis of the amino acid neural tetrapeptide
Sour formicester (BOC-Phe-Arg-OMe) through G-10 column chromatography after purification, molecular weight is 435.4, 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
Measure as 289.3, quasi-molecular ions is 290.3 (M+1)；
Through G-10 column chromatography after purification, molecular weight is 564.45 to four peptide prod Phe-Arg-Trp-Gly, and quasi-molecular ions is
565.45(M+1).
The synthetic method of the amino acid neural tetrapeptide that the present invention is provided, 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),
The pretreatment of alkali protease (Alcalase), tertiary butyloxycarbonyl tryptophan glycine second fat (BOC-Trp-Gly-OEt) remove-insurance
Shield, tertbutyloxycarbonyl phenylalanine arginine tryptophan glycine second fat (BOC-Phe-Arg-Trp-Gly-OEt) synthesis, tertiary fourth
Oxygen carbonyl phenylalanine arginine tryptophan glycine second fat (BOC-Phe-Arg-Trp-Gly-OEt) deprotection, phenylalanine
Arginine tryptophan glycine second fat (Phe-Arg-Trp-Gly-OEt) deprotection, peptide prod are isolated and purified, and the present invention is non-aqueous
Enzymatic clarification amino acid neural tetrapeptide Phe-Arg-Trp-Gly in medium, be in corticotropin (ACTH) activity
The tetrapeptide of center portion position, selects organic solvent dimethylformamide (DMF) during two peptide symthesis, selects organic solvent uncle during four peptide symthesis
Butanol, and using triethylamine cosolvent and appropriate increase water content is added in non-aqueous organic solvent, increase hydrophilic amino acid
Solubility, solve the Utilizing question of hydrophilic amino acid.Improve the availability of hydrophilic amino acid, enzymatic clarification reaction condition
Gently, stereocpecificity is strong, without Side chain protective group, several nothing side reaction, than conventionally used chemical synthesis environmental protection, does not produce
Chiral molecules, without splitting further, improves yield, and optimizes reaction condition.
Description of the drawings
Fig. 1 is the flowchart of the synthetic method of amino acid neural tetrapeptide provided in an embodiment of the present invention；
Fig. 2 is impact schematic diagram of the water content provided in an embodiment of the present invention to dipeptides synthetic yield；
Fig. 3 is the impact schematic diagram for water content to tetrapeptide synthetic yield provided in an embodiment of the present invention；
Fig. 4 is impact schematic diagram of the temperature provided in an embodiment of the present invention to dipeptides synthetic yield；
Fig. 5 is impact schematic diagram of the temperature provided in an embodiment of the present invention to tetrapeptide synthetic yield；
Fig. 6 is impact schematic diagram of the pH provided in an embodiment of the present invention to dipeptides synthetic yield；
Fig. 7 is impact schematic diagram of the pH provided in an embodiment of the present invention to tetrapeptide synthetic yield；
Fig. 8 is two peptide prods tertbutyloxycarbonyl phenylalanine arginine formicester (BOC-Phe- provided in an embodiment of the present invention
Arg-OMe) the HPLC figure through G-10 column chromatography after purification；
Fig. 9 is two peptide prod tertbutyloxycarbonyl phenylalanine arginine formicesters of synthesis provided in an embodiment of the present invention
(BOC-Phe-Arg-OMe) mass spectrogram through G-10 column chromatography after purification；
Figure 10 is two peptide prod tryptophan glycine second fat (Trp-Gly-OEt) warps of synthesis provided in an embodiment of the present invention
Cross G-10 column chromatography HPLC figure after purification；
Figure 11 is that the two peptide prod Trp-Gly-OEt for synthesizing provided in an embodiment of the present invention are purified through G-10 column chromatography
Mass spectrogram afterwards；
Figure 12 is the four peptide prod Phe-Arg-Trp-Gly for synthesis provided in an embodiment of the present invention through G-10 column chromatography
HPLC figure after purification；
Figure 13 is that four peptide prod Phe-Arg-Trp-Gly of synthesis provided in an embodiment of the present invention are pure through G-10 column chromatography
Mass spectrogram after change.
Specific embodiment
In order that the objects, technical solutions and advantages of the present invention become more apparent, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that specific embodiment described herein is not used to only in order to explain the present invention
Limit the present invention.
As shown in figure 1, the present invention is achieved in that a kind of synthetic method of amino acid neural tetrapeptide, amino acid god
Synthetic method through tetrapeptide is comprised the following steps：
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 solution (Tris-HCl buffer), adds chymotrypsin (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 acid terminating 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 to add quality for 0.16g (2ml), Gly-OEt is 0.62g (4mmol), organic solvent dimethylformamide (DMF)
20ml, 1ml triethylamine, a certain amount of tris-HCI buffer (Tris-HCl buffer), add pancreas to coagulate
Galactase (Chymotyrpsin) 2g, 40 DEG C of water-baths vibrate 3 days, take 50 μ l every 8h and add 500 μ l glacial acetic acid terminating reactions,
Generation with Mass Spectrometer Method product；
Step S103, the pretreatment of alkali protease (Alcalase)：Take 2ml alkali protease (Alcalase) and 15ml
Absolute ethyl alcohol is placed in centrifuge tube, and vortex vibration is sufficiently mixed which in 5 minutes, and mixture 3000rpm is centrifuged 10 minutes, alkaline egg
White enzyme (Alcalase) is completely separable with solvent absolute ethyl alcohol, the ethanol on upper strata of inclining, and this step operation is in triplicate；
Step S104, 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-
Phe-Arg-OMe) and tertiary butyloxycarbonyl tryptophan glycine second fat (BOC-Trp-Gly-OEt) organic solvent dimethyl formyl
Amine (DMF), respectively vacuum distillation remove organic solvent dimethylformamide (DMF), then are dissolved with ethyl acetate, and ethyl acetate divides
Not with cold lemon pickling three times, cold sodium acid carbonate is washed twice, then is washed twice with cold water, vacuum distillation, except ethyl acetate, is obtained micro-
Yellow amorphous thing；
Step S105, 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 dissolve, 0 DEG C is shaken
Ether is added after swinging 2-4 hour：Petroleum ether (1:3), must precipitate, centrifugation, add by ether and petroleum ether according to 1:3 ratios are mixed
Close the mixed liquor for constituting and precipitation is washed, obtain tryptophan glycine second fat (Trp-Gly-OEt)；
Step S106, tertbutyloxycarbonyl phenylalanine arginine tryptophan glycine second fat (BOC-Phe-Arg-Trp-
Gly-OEt) synthesize：Take tertbutyloxycarbonyl phenylalanine arginine formicester (BOC-Phe-Arg-OMe) of 2mmol and 4mmol
Tryptophan glycine second fat (Trp-Gly-OEt) adds conical flask, adds organic solvent tert-butyl alcohol 20ml, alkali protease
(Alcalase) 2ml, 1ml triethylamine, a certain amount of Na2CO3-NaHCO3Buffer solution, 37 DEG C of water-baths vibrate 24 hours, every 4h
Take 50 μ l and 500 μ l glacial acetic acid terminating reactions are added, with the generation of Mass Spectrometer Method product；
Step S107, tertbutyloxycarbonyl phenylalanine arginine tryptophan glycine second fat (BOC-Phe-Arg-Trp-
Gly-OEt) deprotection：Tertbutyloxycarbonyl phenylalanine arginine tryptophan glycine second fat (BOC-Phe-Arg-Trp-Gly-
OEt) product TFA (CF3COOH):Methyl phenyl ethers anisole (9:1) just dissolve, after 0 DEG C of vibration 2-4 hour, add ether：Petroleum ether (1:
3), must precipitate, centrifugation, add by ether and petroleum ether according to 1:Precipitation washed by the mixed liquor that the mixing of 3 ratios is constituted, and obtains phenylpropyl alcohol ammonia
Sour arginine tryptophan glycine second fat (Phe-Arg-Trp-Gly-OEt)；
Step S108, phenylalanine arginine tryptophan glycine second fat (Phe-Arg-Trp-Gly-OEt) deprotection：Take
4.4g phenylalanine arginine tryptophan glycine second fat (Phe-Arg-Trp-Gly-OEt), adds 24ml methyl alcohol,
12.5mlNaOH (2mol/L), reacts one hour, adjusts pH to 7 with HCl (1mol/L), pumps methyl alcohol, be diluted with water, filters,
Ice bath is cooled down, and is washed to neutrality, is obtained amino acid neural tetrapeptide (Phe-Arg-Trp-Gly), use Mass Spectrometer Method product；
Step S109, peptide prod are isolated and purified：Post material adopts Sephadex G-10, and chromatographic column is 16mm × 1000mm,
Eluent adopts distilled water, and Detection wavelength is 220nm, and flow velocity is 1.0ml/min, takes the dry post material of 100g Sephadex G-10,
Plus 500ml distilled water immersion is overnight, after decompression bubble removing, post is filled, eluent is balanced overnight, liquid 0.5ml loading layer to be separated
Analysis, collects product peak, is lyophilized to obtain product.
In embodiments of the present invention, the synthetic method of the amino acid neural tetrapeptide detects peptide using high-efficient liquid phase technique (HPLC)
Synthesis purity, the method for quantitative analysis peptide prod are used for the analysis detection of synthetic peptide, using high performance liquid chromatograph, model
The Diamosil chromatographic column of C18,250 × 4.6mm, 220nm DAD UV-detector, 25 DEG C of column temperature, solvent orange 2 A adopt 0.1% 3
The acetonitrile of fluoroacetic acid, using the water containing 0.1% trifluoroacetic acid, flow velocity is 1.0ml/min to solvent B, and concrete gradient is：
In embodiments of the present invention, the synthetic method of the amino acid neural tetrapeptide determines the method mirror of molecular weight using mass spectrum
Determine 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℃；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 the amino acid neural tetrapeptide
10 molecular sieve gel column chromatography for separation, collect product elution liquid peak, secondary repetition loading after rotary evaporation concentration, the wash-out for obtaining
After liquid is concentrated, be lyophilized, the difference assay of container quality itself is deducted using the gross mass of container and product, calculates gained peptide prod
Weight, recycle high-efficient liquid phase technique detection, weight is multiplied by according to peptide prod purity, obtains the weight of pure products, this weight with
The ratio of theoretical yield is exactly ultimate yield.
In embodiments of the present invention, chymotrypsin catalyzes and synthesizes two in organic solvent dimethylformamide (DMF)
During peptide tertbutyloxycarbonyl phenylalanine arginine formicester (BOC-Phe-Arg-OMe), chymotrypsin and organic solvent diformazan
The synthetic system water content that base formamide (DMF) is constituted 1.5%, reaction temperature 40 DEG C, pH be 7.5 when, tertbutyloxycarbonyl
Phenylalanine arginine formicester (BOC-Phe-Arg-OMe) yield highest；
Chymotrypsin catalyzes and synthesizes dipeptides tertiary butyloxycarbonyl tryptophan in organic solvent dimethylformamide (DMF)
During glycine second fat (BOC-Trp-Gly-OEt), the conjunction that chymotrypsin is constituted with organic solvent dimethylformamide (DMF)
Architectonical water content 1.5%, reaction temperature 40 DEG C, pH be 8.0 when, tertiary butyloxycarbonyl tryptophan glycine second fat (BOC-
Trp-Gly-OEt) yield highest；
Alkali protease (Alcalase) catalyzes and synthesizes tetrapeptide tertbutyloxycarbonyl phenylalanine in the organic solvent tert-butyl alcohol
During arginine tryptophan glycine second fat (BOC-Phe-Arg-Trp-Gly-OEt), alkali protease (Alcalase) with organic
Solvent tertiary butanol constitute synthetic system water content 9%, reaction temperature 37 DEG C, pH be 9.0 when, tetrapeptide tertbutyloxycarbonyl benzene
Alanine arginine tryptophan glycine second fat (BOC-Phe-Arg-Trp-Gly-OEt) synthetic yield highest.
In embodiments of the present invention, two peptide prod tertbutyloxycarbonyl benzene of the synthetic method synthesis of the amino acid neural tetrapeptide
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
Measure as 289.3, quasi-molecular ions is 290.3 (M+1)；
Through G-10 column chromatography after purification, molecular weight is 564.45 to four peptide prod Phe-Arg-Trp-Gly, and quasi-molecular ions is
565.45(M+1).
Below in conjunction with the accompanying drawings and specific embodiment is further described to the application principle of the present invention.
3 experimental sections
3.1 experiment material
3.1.1 amino acid and its derivative
BOC-Phe；Arg-OMe.2HCl；BOC-Trp；Gly-OEt.HCl. (gill is purchased from biochemical)
3.1.2 enzyme
Papain (Papain, Merch Germany,
Chymotrypsin (Chymotrypsin), Serva USA,
Trypsase (trypsin), purchased from Beijing Ding Guo biotechnology Co., Ltd (Geview packing)
Alkali protease Alcalase, purchased from Novo-Nordisk (Denmark)
3.1.3 reagent
Tetrahydrofuran, absolute ethyl 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), NaOH, disodium hydrogen phosphate, sodium dihydrogen phosphate, dimethylformamide
(DMF), watery hydrochloric acid, NaCl, sodium carbonate, sodium acid carbonate, trishydroxymethylaminomethane (Tris), ethyl acetate, isopropanol, lemon
Acid, methyl phenyl ethers anisole, ether, petroleum ether, methyl alcohol, isobutanol, it is pure that tert-butyl alcohol etc. is analysis.
3.1.4 filler is isolated and purified
Chromatographic stuffing SephadexG-10, Pharmacia
3.2 instrument
Power DryLL3000 freeze drier (Heto company),
J2-21M high speed freezing centrifuge (Beckman, the U.S.),
JJ-1 type timing electric mixer (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 box (Shanghai laboratory 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.),
Analytic type high pressure liquid chromatograph (the Waters U.S.),
LM17 type recorder (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), and organic solvent 20ml, 1ml triethylamine, a certain amount of trishydroxymethylaminomethane-hydrochloric acid delay
Liquid (Tris-HCl buffer) is rushed, enzyme 2g is added, 40 DEG C of water-baths are vibrated 3 days, 50 μ l are taken every 8h and added for 500 μ l glacial acetic acid ends
Only react, with the generation of Mass Spectrometer Method product.
(2) synthesis of tertiary butyloxycarbonyl tryptophan glycine second fat (BOC-Trp-Gly-OEt)
It is that 0.16g (2ml), Gly-OEt add quality for 0.62g (4mmol) that BOC-Trp adds quality, organic solvent
20ml, 1ml triethylamine, a certain amount of tris-HCI buffer (Tris-HCl buffer), enzyme 2g is added,
40 DEG C of water-baths are vibrated 3 days, take 50 μ l every 8h and add 500 μ l glacial acetic acid terminating reactions, with the generation of Mass Spectrometer Method product.
3.3.1.2 enzyme' s catalysis of Phe-Arg-Trp-Gly tetrapeptide
(1) pretreatment of enzyme：Take 2ml Alcalase and 15ml absolute ethyl alcohol is placed in centrifuge tube, vortex vibrates 5 minutes
It is sufficiently mixed which, mixture 3000rpm is centrifuged 10 minutes, and enzyme is completely separable with solvent, the ethanol on upper strata of inclining, this step 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), vacuum distillation respectively removes organic molten
Agent, product are dissolved with ethyl acetate again.Ethyl acetate respectively with cold lemon pickling three times, wash twice, then uses by cold sodium acid carbonate
Cold water is washed twice, vacuum 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 dissolve, 0 DEG C of vibration 2-4
Ether is added after hour：Petroleum ether (1:3), must precipitate, centrifugation.Add by ether and petroleum ether according to 1:3 ratios mix structure
Precipitation washed by the mixed liquor for becoming, and 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, tri- second of enzyme 2ml, 1ml
Amine, a certain amount of Na2CO3-NaHCO3Buffer solution, 37 DEG C of water-baths vibrate 24 hours, take 50 μ l every 4h and add 500 μ l glacial acetic acids
Terminating reaction, with the generation of Mass Spectrometer Method product.
(5) tertbutyloxycarbonyl phenylalanine arginine tryptophan glycine second fat (BOC-Phe-Arg-Trp-Gly-OEt)
Same 3.3.1.2 (3) tertiary butyloxycarbonyl tryptophan glycine second fat (BOC-Trp-Gly-OEt) the dipeptides remove-insurance of tetrapeptide deprotection method
The method of shield, obtains phenylalanine arginine tryptophan glycine second fat (Phe-Arg-Trp-Gly-OEt).
(6) phenylalanine arginine tryptophan glycine second fat (Phe-Arg-Trp-Gly-OEt) deprotection, takes on 4.4g
Step (5) phenylalanine arginine tryptophan glycine second fat (Phe-Arg-Trp-Gly-OEt), adds 24ml methyl alcohol,
12.5mlNaOH (2mol/L), reacts one hour, adjusts pH7 with HCl (1mol/L), pumps methyl alcohol, be diluted with water, and filters, ice bath
Cooling, washes 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, Detection wavelength：220nm,
Flow velocity：1.0ml/min；
The dry post material of 100g Sephadex G-10 is taken, plus 500ml distilled water immersion is overnight, after decompression bubble removing, fill post.Wash
De- liquid is balanced overnight.Liquid 0.5ml loading chromatography to be separated.The separation for being mainly used in synthesizing dipeptides, tetrapeptide and its derivative is pure
Change.Product peak is collected, is lyophilized to obtain product.
3.3.3 the analysis of peptide prod
High-efficient liquid phase technique (HPLC) detects peptide symthesis purity：
The method of quantitative analysis peptide prod, detects for the analysis of synthetic peptide,
High performance liquid chromatograph：Waters
Chromatographic column：C18,250 × 4.6mm, Diamosil
Detector：DAD UV-detector, 220nm
Column temperature：25℃
Elution requirement：
Solvent orange 2 A:The acetonitrile of 0.1% trifluoroacetic acid
Solvent B:The water of 0.1% trifluoroacetic acid
Flow velocity:1.0ml/min.
3.3.4 the identification of peptide prod
The method that the identification of product mainly determines molecular weight using mass spectrum., because of its mild condition, catalysis is specially for enzymatic peptide symthesis
One property is strong, therefore the basic fracture without the internal covalent bond of amino acid, and due to the use of N-terminal protected amino acid being substrate, substantially
The generation of the accessory substance contrary with target product amino acid sequence is avoided, therefore the method by determining molecular weight can just 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℃；Sweep limits：100-800a.m.u.(atomic mass units).
3.3.5 calculation of yield
The peptide prod of synthesis Sephedex G-10 molecular sieve gel column chromatography for separation, collects product elution liquid peak, rotation
Secondary repetition loading after evaporation and concentration, after the eluent for obtaining is concentrated, is lyophilized, using the difference assay (gross mass of container and product
Deduct the quality of container itself) weight of products therefrom is calculated, high-efficient liquid phase technique detection is recycled, weight is multiplied by according to its purity
Amount, obtains the weight of pure products, and the ratio of this weight and theoretical yield is exactly ultimate yield.
4 results and discussion
4.1 enzymes and the selection of organic solvent
(1) synthesis of enzymatic dipeptides
Different enzyme used in different organic solvents, operating procedure square method 3.3.1.1, solution ph are all used
Tris-HCl buffer solution adjust, water content and using organic solvent be shown in Table 1, the results are shown in Table 1.
1 enzymatic of table, 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), trypsase (Trypsin)
Chymotrypsin (Chymotyrpsin), DMF (dimethylformamide)
Using the synthesis of four kinds of enzymatic dipeptides, as can be seen from Table 1, generally, chemo-enzymatic peptide synthetic yield is in 20-
40% or so, but to catalyze and synthesize yield in DMF higher for chymotrypsin, reaches more than 60%, illustrates that this system is suitable for
The synthesis of dipeptides.This is because the aromatic amino acid such as Phe and Trp is the hydrolysis substrate of chymotrypsin, with pancreas curdled milk egg
White enzyme active center matches, and the activated centre of chymotrypsin is entered beneficial to aromatic amino acid, therefore synthetic yield meeting
Highest.And active sites of other enzymes due to substrate with enzyme not bery coincide, so yield is relatively low.
(2) tertbutyloxycarbonyl phenylalanine arginine tryptophan glycine second fat (BOC-Phe-Arg-Trp-Gly-OEt)
The enzyme' s catalysis of tetrapeptide
The synthesis of enzymatic tetrapeptide, square method 3.3.1.2 (4), the sweet ammonia of tertbutyloxycarbonyl phenylalanine arginine tryptophan
The enzyme' s catalysis of sour second fat (BOC-Phe-Arg-Trp-Gly-OEt) tetrapeptide the results are shown in Table 2.
2 enzymatic of table, four peptide symthesis
Alcalase synthesizes tetrapeptide yield highest in the tert-butyl alcohol as seen from Table 2.Alcalase be from hay bacillus
A kind of protease for extracting, main component hay bacillus (Subtilisin) serine protease, is one of bacterialprotease, it
Good vigor can be kept in alcoholic solution.In four peptide symthesis, four peptide symthesis are catalyzed in the tert-butyl alcohol using Alcalase, by
Hydrophilic in t-butanol solvent, so two peptide substrates dissolubilities are higher than other organic solvent solubility, triethylamine can also promote bottom
The dissolving of thing, in addition Alcalase is higher for the synthetic yield containing hydrophilic amino acid.As solvent hydrophily strengthens, pawpaw
Protease, trypsase and chymotrypsin vigor in the tert-butyl alcohol is lost a lot, and must be added in other solvents
A large amount of water make substrate dissolve very well, and otherwise yield is very low.Meanwhile, two peptide substrates do not meet the activated centre of chymotrypsin
Structure, so yield is not very high.By experiment, the enzyme Alcalase played a decisive role by peptide symthesis is have found.
In enzymatic peptide symthesis, the selection of organic solvent is also critically important, this is because replacing water phase with organic solvent, puts down
Weighing is carried out to peptide symthesis direction, after peptide is generated, is diffused in organic solvent, and reaction can just be smoothed out.Enzyme is also only some
Just vibrant in organic solvent, in other solvents, vigor is very little or none.Hydrophily for different solvents and polarity situation,
Synthesis dipeptides and tetrapeptide have selected different organic solvents respectively, and from the point of view of experimental result, these organic solvents are closed to chemo-enzymatic peptide
Yield is become to have a certain impact, wherein chymotrypsin yield in dimethylformamide is higher, and Alcalase is in the tert-butyl alcohol
Middle yield is higher.Organic solvent is selected to first have to consider its hydrophily that hydrophilic organic solvent can dissolve hydrophilic amino
Acid, increases concentration of substrate and can just improve yield, and DMF and the tert-butyl alcohol are all miscible with water.Another Consideration is organic solvent
Boiling point, need vacuum distillation or rotary evaporation when product is processed, if boiling point is too high, product separate highly difficult.DMF and tertiary fourth
Alcohol boiling point is at 100 DEG C or so.In addition it is also considered that organic solvent toxicity, selects the less organic solvent of toxicity, DMF as far as possible
Toxicity very little with the tert-butyl alcohol.
Impact of the 4.2 system water contents to peptide symthesis
Fig. 2 is impact schematic diagram of the water content to dipeptides synthetic yield, and Fig. 3 is impact of the water content to tetrapeptide synthetic yield
Schematic diagram.From figure 2, it is seen that water content is at 1.5%, dipeptides yield reaches highest.As can be seen from Figure 3 water content is at 9%, tetrapeptide
Yield reaches highest.Impact of the Water in Organic Solvents content to enzymatic activity is very important, and the water in usual system is with three kinds
Mode is present：The water combined with enzyme, the combination water with water-insoluble, the water for dissociating in a solvent.Enzyme is catalyzed in organic solvent
There is an optimum moisture content, when water content is higher than most suitable water content, water can compete the binding site of nucleopilic reagent, cause ester bottom
Thing is hydrolyzed, and reduces the yield of target product, and when water content is less than most suitable water content, enzyme conformation is excessively rigid, while solvent
The highly polar necessary water for having captured enzyme, causes the catalysis activity of enzyme to reduce and even inactivates, and only under water content is best suitable for, enzyme divides
The existing certain rigidity of son, has certain flexibility again, and peptide yield could highest.The water content of system is had with the hydrophobicity of solvent
Close, solvent is more hydrophilic, and system water content is more, because solvent is hydrophilic, the ability for fighting for the necessary water of enzyme is stronger, only many
Plus a little water, just can guarantee that the activity of enzyme.This be also water content in Alcalase- tert-butyl alcohol system higher than chymotrypsin-
One of the reason for DMF content of water in system, the hydrophily of the tert-butyl alcohol, are greater than the hydrophily of DMF.In addition, the water in reaction system contains
Amount not only affects the catalysis activity of enzyme, has an effect on the stereoselectivity of enzyme, this is because water changes the flexibility of enzyme molecule, mapping
The selectivity of body and water content have dependence.
The impact of 4.3 reaction temperatures
Fig. 4 is impact schematic diagram of the temperature to dipeptides synthetic yield, and Fig. 5 is that impact of the temperature to tetrapeptide synthetic yield is illustrated
Figure.As can be seen from Figure 4, when reaction temperature is at 40 DEG C, dipeptides yield reaches highest；As can be seen from Figure 5, when reaction temperature is at 37 DEG C
When, tetrapeptide yield reaches highest.Generally improve reaction temperature and can improve enzymatic reaction speed.Temperature is too low, reaction speed
Degree is very slow；But temperature is too high to cause enzyme denaturation, cause enzymatic vigor to be decreased obviously.This study tour reaction temperature is to mesh
The impact of mark peptide symthesis yield.Think in enzyme molecule in temperature is best suitable for, vigor reaches highest.Find in an experiment,
Enzyme optimum temperature in organic solvent is higher than the optimum temperature of water phase, this is because enzyme molecule electrostatic phase in organic solvent
Interaction is greater than the electrostatic interaction in water phase, and molecular rigidity strengthens, and flexible reduction, its heat endurance are improved, and this is also enzyme
An advantage of peptide symthesis is catalyzed in organic solvent.
The impact of 4.4 systems pH
Fig. 6 is impact schematic diagram of the pH to dipeptides synthetic yield, and Fig. 7 is impact schematic diagram of the pH to tetrapeptide synthetic yield.
As seen from Figure 6, when pH is 7.5, tertbutyloxycarbonyl phenylalanine arginine formicester (BOC-Phe-Arg-OMe) yield reaches
Highest, when pH is 8.0, tertiary butyloxycarbonyl tryptophan glycine second fat (BOC-Trp-Gly-OEt) synthetic yield reaches highest；
As seen from Figure 7, when pH is 9.0, tetrapeptide tertbutyloxycarbonyl phenylalanine arginine tryptophan glycine second fat (BOC-Phe-
Arg-Trp-Gly-OEt) synthetic yield reaches highest.Enzyme also has optimal pH scope in organic solvent, difference compared with water
Less.PH can affect the dissociated state of substrate molecule, can also affect the dissociated state of enzyme molecule, optimal pH and the dissociation of the two
There is relation, under optimal pH, the activated centre molecule of substrate molecule and enzyme is in most easy bonding state, with this understanding enzyme activity
Power highest, can combine to form compound intermediate well, be conducive to peptide symthesis.In addition, pH affects enzyme molecule in organic solvent
The dissociation of some groups, is that the conformation of enzyme changes so as to which optimal pH changes, and also affects the yield of peptide symthesis.
The analysis of 4.5 peptide prods and identification
Fig. 8 is shown that the two peptide prod tertbutyloxycarbonyl phenylalanine arginine formicester (BOC-Phe-Arg- for synthesizing
OMe) the HPLC figure through G-10 column chromatography after purification.
Fig. 9 is that two peptide prods tertbutyloxycarbonyl phenylalanine arginine formicester (BOC-Phe-Arg-OMe) of synthesis are passed through
G-10 column chromatography mass spectrogram after purification, molecular weight are 435.4, and on mass spectrogram, quasi-molecular ions is 436.4 (M+1).
Figure 10 be synthesis two peptide prods tryptophan glycine second fat (Trp-Gly-OEt) through G-10 column chromatography after purification
HPLC figure.
Figure 11 be synthesis two peptide prods tryptophan glycine second fat (Trp-Gly-OEt) through G-10 column chromatography after purification
Mass spectrogram, molecular weight be 289.3, on mass spectrogram quasi-molecular ions be 290.3 (M+1).
Figure 12 is HPLC figure of the four peptide prod Phe-Arg-Trp-Gly of synthesis through G-10 column chromatography after purification.
Figure 13. it is shown that mass spectrum of the four peptide prod Phe-Arg-Trp-Gly for synthesizing through G-10 column chromatography after purification
Figure, molecular weight are 564.45, and on mass spectrogram, quasi-molecular ions is 565.45 (M+1).
5 conclusions
In the peptide symthesis research with amino acid neural tetrapeptide Phe-Arg-Trp-Gly as target, investigate to peptide symthesis
Influential many factors, have found optimal enzyme to be catalyzed the synthesis of peptide first, secondly find organic solvent to peptide symthesis
Yield also has an impact, and have found suitable organic solvent DMF and the tert-butyl alcohol.Change system water content, pH and reaction in an experiment
Temperature is constructed optimum response model, makes the yield of dipeptides arrive separately at 61.25%, 85.6% finding optimum reaction condition,
Tetrapeptide yield also reaches 68.32%.
This experiment problem to be solved is the Utilizing question of hydrophilic substrates, then first have to consider hydrophilic substrates
Solubility in organic solvent, this is necessary for selecting appropriate organic solvent, selects DMF during two peptide symthesis, selects during four peptide symthesis
The tert-butyl alcohol is selected, both solvents all have good dissolubility to substrate, triethylamine is secondly added, its objective is to increase the molten of substrate
Xie Xing, contributes to the carrying out that reacts.
The synthetic method of amino acid neural tetrapeptide provided in an embodiment of the present invention, including：Tertbutyloxycarbonyl phenylalanine essence
The synthesis of propylhomoserin formicester (BOC-Phe-Arg-OMe), tertiary butyloxycarbonyl tryptophan glycine second fat (BOC-Trp-Gly-OEt)
Synthesis, the pretreatment of alkali protease (Alcalase), tertiary butyloxycarbonyl tryptophan glycine second fat (BOC-Trp-Gly-OEt)
Deprotection, tertbutyloxycarbonyl phenylalanine arginine tryptophan glycine second fat (BOC-Phe-Arg-Trp-Gly-OEt) synthesis,
Tertbutyloxycarbonyl phenylalanine arginine tryptophan glycine second fat (BOC-Phe-Arg-Trp-Gly-OEt) deprotection, phenylpropyl alcohol
Propylhomoserin arginine tryptophan glycine second fat (Phe-Arg-Trp-Gly-OEt) deprotection, peptide prod are isolated and purified, and the present invention exists
Enzymatic clarification amino acid neural tetrapeptide Phe-Arg-Trp-Gly in non-aqueous media, is that corticotropin (ACTH) is living
Property centre tetrapeptide, during two peptide symthesis select organic solvent dimethylformamide (DMF), select during four peptide symthesis organic molten
The agent tert-butyl alcohol, and using triethylamine cosolvent and appropriate increase water content is added in non-aqueous organic solvent, increase hydrophilic ammonia
The solubility of base acid, solves the Utilizing question of hydrophilic amino acid.
Presently preferred embodiments of the present invention is the foregoing is only, not in order to limit the present invention, all essences in the present invention
Any modification, equivalent and improvement that is made within god and principle etc., should be included within the scope of the present invention.
Claims (6)
1. a kind of synthetic method of amino acid neural tetrapeptide, it is characterised in that the synthetic method of the amino acid neural tetrapeptide includes
Following steps：
The synthesis of tertbutyloxycarbonyl phenylalanine arginine formicester：It is 0.53g that tertbutyloxycarbonyl phenylalanine adds quality, smart ammonia
It is 1.04g that sour formicester adds quality, organic solvent dimethylformamide 20ml, 1ml triethylamine, trishydroxymethylaminomethane-salt
Acid buffer, adds chymotrypsin 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 to terminate instead
Should, with the generation of Mass Spectrometer Method product；
The synthesis of tertiary butyloxycarbonyl tryptophan glycine second fat：It is that 0.16g, Gly-OEt addition quality is that BOC-Trp adds quality
0.62g, organic solvent dimethylformamide 20ml, 1ml triethylamine, tris-HCI buffer, add pancreas to coagulate
Galactase 2g, 40 DEG C of water-baths vibrate 3 days, take 50 μ l every 8h and add 500 μ l glacial acetic acid terminating reactions, use Mass Spectrometer Method product
Generation；
The pretreatment of alkali protease：Take 2ml alkali protease and 15ml absolute ethyl alcohol is placed in centrifuge tube, vortex vibrates 5 points
Clock is sufficiently mixed which, and mixture 3000rpm is centrifuged 10 minutes, and alkali protease is completely separable with solvent absolute ethyl alcohol, inclines
The ethanol on upper strata, this step operation is in triplicate；
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
Other vacuum distillation removes organic solvent dimethylformamide, then is dissolved with ethyl acetate, and ethyl acetate is respectively with cold lemon pickling
Three times, cold sodium acid carbonate is washed twice, then is washed twice with cold water, vacuum distillation, except ethyl acetate, obtains slightly yellow amorphous article；
Tertiary butyloxycarbonyl tryptophan glycine second fat deprotection：Tertiary butyloxycarbonyl tryptophan glycine second fat product TFA benzene first
Ether=9:1 just dissolves, and adds ether after 0 DEG C of vibration 2-4 hour：Petroleum ether=1:3, must precipitate, centrifugation, add by ether
With petroleum ether according to 1:Precipitation washed by the mixed liquor that the mixing of 3 ratios is constituted, and obtains tryptophan glycine second fat；
Tertbutyloxycarbonyl phenylalanine arginine tryptophan glycine second fat synthesizes：Take the tertbutyloxycarbonyl phenylalanine of 2mmol
The tryptophan glycine second fat of arginine formicester and 4mmol adds conical flask, adds organic solvent tert-butyl alcohol 20ml, basic protein
Enzyme 2ml, 1ml triethylamine, Na2CO3-NaHCO3Buffer solution, 37 DEG C of water-baths vibrate 24 hours, take 50 μ l every 4h and add 500 μ l ice
Acetic acid terminating reaction, with the generation of Mass Spectrometer Method product；
Tertbutyloxycarbonyl phenylalanine arginine tryptophan glycine second fat deprotection：Tertbutyloxycarbonyl phenylalanine arginine color
Propylhomoserin glycine second fat product TFA:Methyl phenyl ethers anisole=9:1 just dissolves, and adds ether after 0 DEG C of vibration 2-4 hour：Petroleum ether=
1:3, must precipitate, centrifugation, add by ether and petroleum ether according to 1:Precipitation washed by the mixed liquor that the mixing of 3 ratios is constituted, and obtains phenylpropyl alcohol
Propylhomoserin arginine tryptophan glycine second fat；
Phenylalanine arginine tryptophan glycine second fat deprotection：Take 4.4g phenylalanine arginine tryptophan glycine second
Fat, adds 24ml methyl alcohol, and the NaOH12.5ml of 2mol/L, reaction one hour, the HCl regulation pH with 1mol/L pump first to 7
Alcohol, is diluted with water, and filters, and ice bath is cooled down, and washes to neutrality, obtains amino acid neural tetrapeptide, use Mass Spectrometer Method product；
Peptide prod is isolated and purified：Post material adopts Sephadex G-10, and chromatographic column is 16mm × 1000mm, and eluent is using distillation
Water, Detection wavelength are 220nm, and flow velocity is 1.0ml/min, take the dry post material of 100g SephadexG-10, plus 500ml distillation water logging
Bubble overnight, fills post after decompression bubble removing, and eluent is balanced overnight, liquid 0.5ml loading chromatography to be separated, collects product peak, freezes
Do to obtain product.
2. the synthetic method of amino acid neural tetrapeptide as claimed in claim 1, it is characterised in that the amino acid neural tetrapeptide
Synthetic method detects peptide symthesis purity using high-efficient liquid phase technique, and the method for quantitative analysis peptide prod is used for the analysis inspection of synthetic peptide
Survey, using high performance liquid chromatograph, model C18, the Diamosil chromatographic column of 250 × 4.6mm, 220nm DAD ultraviolet detection
Device, 25 DEG C of column temperature, solvent orange 2 A adopt the water containing 0.1% trifluoroacetic acid using the acetonitrile of 0.1% trifluoroacetic acid, solvent B, and flow velocity is
1.0ml/min.
3. the synthetic method of amino acid neural tetrapeptide as claimed in claim 1, it is characterised in that the amino acid neural tetrapeptide
Synthetic method determines the method identification peptide prod of molecular weight using mass spectrum, and Mass Spectrometry Conditions are as follows：Ionization mode：API–ES；Pole
Property：Positive；Voltage：4000V；Nebulizer pressure：35psig；Dry air-flow：10L/min；Temperature：350℃；Sweep limits：
100-800a.m.u.
4. the synthetic method of amino acid neural tetrapeptide as claimed in claim 1, it is characterised in that the amino acid neural tetrapeptide
The peptide prod Sephedex G-10 molecular sieve gel column chromatography for separation of synthetic method synthesis, collects product elution liquid peak, rotation
Secondary repetition loading after evaporation and concentration, after the eluent for obtaining is concentrated, is lyophilized, and deducts container using gross mass of the container with product
The difference assay of quality itself, calculates the weight of gained peptide prod, recycles high-efficient liquid phase technique detection, is taken advantage of according to peptide prod purity
With weight, the weight of pure products is obtained, the ratio of this weight and theoretical yield is exactly ultimate yield.
5. the synthetic method of amino acid neural tetrapeptide as claimed in claim 1, it is characterised in that chymotrypsin is organic
When catalyzing and synthesizing dipeptides tertbutyloxycarbonyl phenylalanine arginine formicester in solvent dimethylformamide, chymotrypsin with have
Machine solvent dimethylformamide constitute synthetic system water content 1.5%, reaction temperature 40 DEG C, pH be 7.5 when, tertiary fourth oxygen
Carbonyl phenylalanine arginine formicester yield highest；
Chymotrypsin catalyzes and synthesizes dipeptides tertiary butyloxycarbonyl tryptophan glycine second in organic solvent dimethylformamide
During fat, the synthetic system water content that chymotrypsin is constituted with organic solvent dimethylformamide exists in 1.5%, reaction temperature
40 DEG C, pH be 8.0 when, tertiary butyloxycarbonyl tryptophan glycine second fat yield highest；
It is sweet that alkali protease catalyzes and synthesizes tetrapeptide tertbutyloxycarbonyl phenylalanine arginine tryptophan in the organic solvent tert-butyl alcohol
During propylhomoserin second fat, synthetic system water content that alkali protease and the organic solvent tert-butyl alcohol are constituted is in 9%, reaction temperature 37
DEG C, pH be 9.0 when, tetrapeptide tertbutyloxycarbonyl phenylalanine arginine tryptophan glycine second fat synthetic yield highest.
6. the synthetic method of amino acid neural tetrapeptide as claimed in claim 1, it is characterised in that the amino acid neural tetrapeptide
Synthetic method synthesis two peptide prod tertbutyloxycarbonyl phenylalanine arginine formicesters through G-10 column chromatography after purification, molecular weight
For 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；
Through G-10 column chromatography after purification, molecular weight is 564.45 to four peptide prod Phe-Arg-Trp-Gly, and quasi-molecular ions is
565.45.
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| CN101518654A (en) * | 2009-03-26 | 2009-09-02 | 广东医学院 | Method for synthesizing novel preparation capable of carrying glucocorticoid to be used as inflammation targeted drug |
| CN101570570A (en) * | 2009-06-03 | 2009-11-04 | 兰州大学 | Synthetic method for somatostatin |
| CN101947310A (en) * | 2004-10-27 | 2011-01-19 | 丹佛大学 | Thyroliberin analog and associated method |
| CN103276034A (en) * | 2013-05-17 | 2013-09-04 | 吉林化工学院 | Precursor dipeptide for synthesizing amino acid neuropeptide by enzyme method |
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|---|---|---|---|---|
| CN101947310A (en) * | 2004-10-27 | 2011-01-19 | 丹佛大学 | Thyroliberin analog and associated method |
| CN101518654A (en) * | 2009-03-26 | 2009-09-02 | 广东医学院 | Method for synthesizing novel preparation capable of carrying glucocorticoid to be used as inflammation targeted drug |
| CN101570570A (en) * | 2009-06-03 | 2009-11-04 | 兰州大学 | Synthetic method for somatostatin |
| CN103276034A (en) * | 2013-05-17 | 2013-09-04 | 吉林化工学院 | Precursor dipeptide for synthesizing amino acid neuropeptide by enzyme method |
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