CN106749529A - A kind of preparation method of Argireline and products thereof - Google Patents
A kind of preparation method of Argireline and products thereof Download PDFInfo
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- CN106749529A CN106749529A CN201710007496.9A CN201710007496A CN106749529A CN 106749529 A CN106749529 A CN 106749529A CN 201710007496 A CN201710007496 A CN 201710007496A CN 106749529 A CN106749529 A CN 106749529A
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- C07—ORGANIC CHEMISTRY
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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Abstract
The present invention provides a kind of preparation method of Argireline, and methods described comprises the following steps：(1) Fmoc Arg (pbf) 2 Chlorotrityl Chloride Resin are synthesized；(2) Fmoc Glu (OtBu) Glu (OtBu) Met Gln (Trt) Arg (pbf) Arg (pbf) 2 Chlorotrityl Chloride Resin are synthesized；(3) Ac Glu (OtBu) Glu (OtBu) Met Gln (Trt) Arg (pbf) Arg (pbf) 2 Chlorotrityl Chloride Resin are synthesized；(4) Ac Glu (OtBu) Glu (OtBu) Met Gln (Trt) Arg (pbf) Arg (pbf) OH is synthesized；And (5) step (4) to obtain product is cut, separates, amidation process and purifying obtain Argireline product.Methods described had both overcome the low problem of existing classical synthesis in solid state Argireline resin high cost, carrying capacity; also overcome liquid phase synthesis Argireline process complexity, the shortcoming that synthesis cycle is long, efficiency is low; with synthesis low cost, combined coefficient be high, the simple advantage of synthetic method, can be used for scale and be combined to Argireline.
Description
Technical field
The invention belongs to peptide synthesis technology field, and in particular to a kind of method for synthesizing Argireline by fragment method.
Background technology
Argireline is a kind of containing six active peptides of amino acid, and its sequence is Ac-Glu-Glu-Met-Gln-Arg-
Arg-NH2, the bioactivity with skin wrinkle is widely used in various senior cosmetics.
At present, the synthetic method of Argireline mainly has solid-phase synthesis and liquid phase synthesizing method.What solid-phase synthesis were used
Fmoc-AM Resin and Fmoc-MBHA Resin are expensive, and resin substitution value is smaller, cause Argireline synthesis in solid state into
This is higher.Although the liquid phase synthesizing method prices of raw materials are substantially reduced, liquid phase synthesizing method building-up process is numerous and diverse, and the cycle is long, efficiency
It is low, purify high cost.Argireline reduces production cost as cosmetic material, and improve production efficiency simplifies grinding for synthesis technique
Study carefully significant.
Therefore, the technology that developing a kind of method of high-efficiency and low-cost synthesis Argireline turns into this area urgent need solution is asked
Topic.
The content of the invention
Present invention solves the technical problem that being to provide, a kind of synthesis low cost, combined coefficient be high, synthetic method is simply excellent
Point, can be used for the method that scale is combined to Argireline, which overcome existing classical synthesis in solid state Argireline resin high cost, carrying capacity
Low problem, also overcomes liquid phase synthesis Argireline process complexity, the shortcoming that synthesis cycle is long, efficiency is low.
Specifically, the invention discloses a kind of synthetic method of Argireline, by 2-Chlorotrityl Ch
Loride Resin synthesize Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-OH all risk insurances
Bluff piece section peptide, then with full guard fragment peptide Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Ar g (pbf)-Arg
(pbf)-OH and hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus, 1- hydroxyls azimidobenzene, ammonium chloride, N, N ' -
Diisopropylethylamine synthesizes Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH2, then
Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH2 deprotection bases are obtained into six victory
Peptide.
More particularly, the present invention is achieved through the following technical solutions.
A kind of preparation method of Argireline, methods described comprises the following steps：
(1) synthesis Fmoc-Arg (pbf) -2-Chlorotrityl Chloride Resin；
(2) Fmoc-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf) -2- is synthesized
Chlorotrityl Chloride Resin；
(3) Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf) -2- is synthesized
Chlorotrityl Chloride Resin；
(4) Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-OH is synthesized；And
(5) step (4) is obtained into product to be cut, separate and purifying obtains Argireline product.
Wherein described step (1) be by 2-Chlorotrityl Chloride Resin with dichloromethane it is swelling after, add
Dichloromethane, Fmoc-Arg (pbf)-OH, N, N '-diisopropylethylamine react and obtain Fmoc-Arg (pbf) -2-
Chlorotrityl Chloride Resin；
Wherein described step (2) is Fmoc-Arg (the pbf) -2-Chlorotrityl Chloride for obtaining step (1)
The de- Fmoc of Resin twice, add DMF, Fmoc-Arg (pbf)-OH, 1- hydroxyls azimidobenzene, the nitrogen of benzo three
Azoles-N, N, N ', N '-tetramethylurea tetrafluoro boric acid, N, N '-diisopropylethylamine react and obtain Fmoc-Arg (pbf)-Arg
(pbf)-2-Chlorotrityl Chloride Resin；Then according to methods described Fmoc-Arg (pbf)-Arg (pbf)-
Fmoc-Gln (Trt)-OH, Fmoc-Met-OH, Fmoc-Glu are sequentially connected on 2-Chlorotrityl Chloride Resin
(OtBu)-OH, Fmoc-Glu (OtBu)-OH, obtain Fmoc-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg
(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin。
Wherein described step (3) is Fmoc-Glu (OtBu)-Glu (the OtBu)-Met-Gln for obtaining step (2)
(Trt) the de- Fmoc- of-Arg (pbf)-Arg (pbf) -2-Chlorotrityl Chloride Resin twice, add dichloromethane
Alkane, acetic anhydride, N, the mixed liquor of N '-diisopropylethylamine carry out reacting to obtain Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln
(Trt)-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin。
Reaction described in the step (1), step (2) and step (3) is the stirring at normal temperature 0.5-2 under nitrogen protection
Hour；It is to be with the volume ratio of piperidines and N,N-dimethylformamide that Fmoc is taken off in the step (1), step (2) and step (3)
1:The de- Fmoc of 4 mixed liquor；Dichloromethane, acetic anhydride, N, N '-diisopropylethylamine volume ratio 9 are added in preferred steps (4):
1:0.5 mixed liquor.
Wherein described step (4) is by Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg
(pbf) cutting liquid is added in -2-Chlorotrityl Chloride Resin carries out cleavage reaction by separating, purifying, dry
Obtain Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-OH；
Wherein step (4) described separation, purifying, drying steps are that, by the product filtering after cutting, filtrate uses twice saturation
Brine It 3 times, then adds the saturated aqueous common salt isometric with cutting liquid, is with saturated sodium bicarbonate solution tune pH
7.0,35 DEG C are evaporated off organic solvent and separate out precipitation, filtering, and precipitation uses pure water 3 times, and 40~45 DEG C dry
Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-OH。
Wherein described step (5) is by Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg
(pbf)-OH DMFs dissolve, and add hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus, 1-
Hydroxyl azimidobenzene, ammonium chloride, N, N '-diisopropylethylamine react obtaining reaction solution, are processed, separated, purified, dried
Do to obtain Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH2；Then to Ac-Glu
(OtBu) cutting liquid is added to be cut in-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH2, afterwards
Isolated crude product, it is purified, be dried to obtain product.
Through filtering after wherein described step (5) cutting, by filtrate addition cold diethyl ether, precipitation is separated out, obtain Argireline
Crude product, the inverted chromatogram purification of Argireline crude product, freeze-drying, obtains Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2 products.
Wherein the step (4) cutting liquid is trifluoroethanol and the volume ratio of acetic acid, dichloromethane is 2:0.75:
7.25 mixed liquor；Preferred steps (5) described cutting liquid is that mass percent composition is trifluoroacetic acid 83%, phenol 5%, benzene first
Thioether 4%, water 3%, the mixed liquor of tri isopropyl silane 5%.
2-chlorotrityl chloride resin and Fmoc-Arg (pbf)-OH, N, N '-two wherein in step (1)
The mol ratio of wopropyl ethyl amine is 1:1:4.
In described step (2), 2-chlorotrityl chloride resin and Fmoc-Arg (pbf)-OH, 1- hydroxyls
Base azimidobenzene, BTA-N, N, N ', N '-tetramethylurea tetrafluoro boric acid, N, the mol ratio 1 of N '-diisopropylethylamine:
2:(2.5~3):(2.5~3):(2.5~3).
In described step (2), Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg
(pbf)-OH and hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus, 1- hydroxyls azimidobenzene, ammonium chloride, N, N ' -
The mol ratio 1 of diisopropylethylamine:3:3:(3~5):(3~5).
The present invention also provides the Argireline product that the preparation method is prepared, and its purity is higher than 95%.
More particularly, the present invention provides a kind of solid phase fragment synthetic method of Argireline, comprises the steps：
A kind of synthetic method of Argireline, it is characterised in that it is made up of following step：
(1) Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-OH is synthesized
1. Fmoc-Arg (pbf) -2-Chlorotrityl Chloride Resin are synthesized
By 2-Chlorotrityl Chloride Resin with after swelling 15 minutes of dichloromethane, add dichloromethane,
Fmoc-Arg (pbf)-OH, N, N '-diisopropylethylamine, under nitrogen protection, stirring at normal temperature 1.5 hours obtains Fmoc-Arg
(pbf)-2-Chlorotrityl Chloride Resin；
2. Fmoc-Arg (pbf)-Arg (pbf) -2-Chlorotrityl Chloride Resin are synthesized
By Fmoc-Arg (pbf) -2-Chlorotrityl Chloride Resin piperidines and N,N-dimethylformamide
Volume ratio be 1:The de- Fmoc- of 4 mixed liquor twice, adds DMF, Fmoc-Ar g (pbf)-OH, 1- hydroxyls
Base azimidobenzene, BTA-N, N, N ', N '-tetramethylurea tetrafluoro boric acid, N, N '-diisopropylethylamine, in nitrogen protection
Under, stirring at normal temperature 2 hours obtains Fmoc-Arg (pbf)-Arg (pbf) -2-Chlorotrityl Chloride Resin；
3. Fmoc-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf) -2-Chlor is synthesized
otrityl Chloride Resin
According to step method 2. to Fmoc-Arg (pbf)-Arg (pbf) -2-Chlorotrityl Chloride Resi
Fmoc-Gln (Trt)-OH, Fmoc-Met-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Glu (OtBu)-OH are sequentially connected on n,
Obtain Fmoc-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (p bf) -2-Chlorotrityl
Chloride Resin；
4. Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf) -2- is synthesized
Chlorotr ityl Chloride Resin
By Fmoc-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf) -2-
Chlorotrit yl Chloride Resin piperidines is 1 with the volume ratio of N,N-dimethylformamide:The de- Fm of 4 mixed liquor
Oc- twice, adds dichloromethane, acetic anhydride, N, N '-diisopropylethylamine volume ratio 9:1:0.5 mixed liquor, in nitrogen protection
Under, stirring at normal temperature 30 minutes obtains Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf) -2-
Chlorotrityl Chloride Resin；
5. cutting process
To Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf) -2-
Cutting liquid, stirring at normal temperature 1.5 hours, filtering, by filtrate saturated common salt are added in Chlorotrityl Chloride Resin
Water washing 3 times, then adds the saturated aqueous common salt isometric with cutting liquid, and it is 7.0 to adjust PH with saturated sodium bicarbonate solution, rotation
Turn 35 DEG C of evaporimeter organic solvent is evaporated off and precipitation is separated out, filter, precipitation uses pure water 3 times, and 40~45 DEG C dry to obtain Ac-
Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-OH；
Above-mentioned cutting liquid is trifluoroethanol and the volume ratio of acetic acid, dichloromethane is 2:0.75:7.25 mixed liquor；
(2) Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH2 is synthesized
By Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-OH N, N- diformazans
Base formyl amine solvent, adds hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus, 1- hydroxyls azimidobenzene, chlorination
Ammonium, N, N '-diisopropylethylamine, stirring reaction 3 hours add 2 times of saturated aqueous common salts of volume and separate out precipitation to reaction solution,
Filtering, precipitation uses pure water 3 times, and 40~45 DEG C dry to obtain Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg
(pbf)-Arg(pbf)-NH2；
(3) synthesis Argireline Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2
Cut to being added in Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH2
Liquid, every gram of Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH2 cutting liquid 3~
5mL, is stirred at room temperature 1.5~2.5 hours, filtering, by filtrate addition cold diethyl ether, separates out precipitation, obtains Argireline crude product, six victory
The inverted chromatogram purification of peptide crude product, freeze-drying, obtain Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2；
Above-mentioned cutting liquid be mass percent composition for trifluoroacetic acid 83%, phenol 5%, thioanisole 4%, water 3%,
The mixed liquor of tri isopropyl silane 5%.
Wherein, described step (1) 1. in, 2-chlorotrityl chloride resin and Fmoc-Arg
(pbf)-OH, N, the mol ratio of N '-diisopropylethylamine is 1:1:4.
Wherein, described step (1) 2. in, 2-chlorotrityl chloride resin and Fmoc-Arg
(pbf)-OH, 1- hydroxyls azimidobenzene, BTA-N, N, N ', N '-tetramethylurea tetrafluoro boric acid, N, N '-diisopropyl second
The mol ratio 1 of amine:2:2.5~3:2.5~3:2.5~3.
Wherein, described step (1) 3. in, 2-chlorotrityl chloride resin and Fmoc-Gln
(Trt)-OH, Fmoc-Met-OH, Fmoc-Glu (OtBu)-OH, the mol ratio of Fmoc-Glu (OtBu)-OH are 1:2,2-
Chlorotrityl chloride resin and 1- hydroxyls azimidobenzene, BTA-N, N, N ', N '-tetramethylurea tetrafluoro
Boric acid, N, the mol ratio 1 of N '-diisopropylethylamine:2.5~3:2.5~3:2.5~3.
Wherein, described step (1) 5. in, obtain Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg
(pbf) method of-Arg (pbf)-OH is, by cutting liquid 2 times of saturated common salt water washings of volume 3 times, then to add and cut
The isometric saturated aqueous common salt of liquid is cut, it is 7.0 to adjust PH with saturated sodium bicarbonate solution, and 35 DEG C of Rotary Evaporators are evaporated off organic solvent
And precipitation is separated out, and filtering, precipitation uses pure water 3 times, 40~45 DEG C of drying.
Wherein, in described step (2), Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg
(pbf)-OH and hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus, 1- hydroxyls azimidobenzene, ammonium chloride, N, N ' -
The mol ratio 1 of diisopropylethylamine:3:3:3~5:3~5.
Wherein, in described step (2), Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg is obtained
(pbf) method of-Arg (pbf)-NH2 is to reaction solution 2 times of saturated aqueous common salts of volume of addition and separates out precipitation, and filtering is precipitated
With pure water 3 times, 40~45 DEG C of drying.
The present invention has the beneficial effect that：
The present invention has synthesized six victory of full guard using more cheap 2-Chlorotrityl Chloride Resin first
Fragments of peptides Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-OH, then by full guard
Argireline fragment c-terminus hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus, 1- hydroxyls azimidobenzene, chlorination
Ammonium, N, the amidatioon in N,N-dimethylformamide solution of N '-diisopropylethylamine obtain Ac-Glu (OtBu)-Glu (OtBu)-
Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH2, then by Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-
Arg (pbf)-Arg (pbf)-NH2 Deprotections obtain Argireline Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2。
Instant invention overcomes Fmoc-AM Resin and Fmoc-MBHA that traditional Argireline solid-phase synthesis are used
Resin is expensive, and resin substitution value is smaller, causes the relatively costly problem of Argireline synthesis in solid state, also overcomes six victory
Peptide liquid phase synthesizing method building-up process is numerous and diverse, and the cycle is long, and efficiency is low, purifies the problem of high cost, is that Argireline large-scale production is carried
For a kind of process it is simple, production cost is relatively low, easy purification, the synthetic method of efficiency high.
The inventive method had both overcome the low problem of existing classical synthesis in solid state Argireline resin high cost, carrying capacity, also gram
Taken that liquid phase synthesis Argireline process is complicated, the shortcoming that synthesis cycle is long, efficiency is low, with synthesis low cost, combined coefficient it is high,
The simple advantage of synthetic method, can be used for scale and is combined to Argireline.
Brief description of the drawings
Fig. 1 is the mass spectrogram of the Argireline of the synthesis of embodiment 1.
Fig. 2 is the liquid chromatogram of the Argireline of the synthesis of embodiment 1, wherein (a) is liquid chromatogram, (b) is logical treatment
Road.
Specific embodiment
The present invention is described in more detail with reference to the accompanying drawings and examples, but the invention is not restricted to these embodiments.
Below to specification in write a Chinese character in simplified form and illustrate：
2-chlorotrityl chloride Resin, its entitled 2- chlorine trityl chloride resin
Fmoc：9- tablet held before the breast by officials methylene oxygen carbonyls
Pbf, OtBu, Trt are protection groups, and title is respectively 2,2,4,6,7- pentamethyl Dihydrobenzofuranes -5- sulphonyl
Base, tert-butoxy, trityl.
The present invention provides a kind of preparation method of Argireline, it is characterised in that methods described comprises the following steps：(1) close
Into Fmoc-Arg (pbf) -2-Chlorotrityl Chloride Resin；(2) Fmoc-Glu (OtBu)-Glu is synthesized
(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin；(3) synthesize
Ac-Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride
Resin；(4) Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-OH is synthesized；And (5)
Step (4) is obtained into product to be cut, separate and purifying obtains Argireline product.Wherein described step (1) is by 2-
Chlorotrityl Chloride Resin with dichloromethane it is swelling after, add dichloromethane, Fmoc-Arg (pbf)-OH, N,
N '-diisopropylethylamine react and obtains Fmoc-Arg (pbf) -2-Chlorotrityl Chloride Resin.The conjunction
It is the result of inventor's creative work into method, Fmoc-Arg (pbf) -2-Chlorotrityl Chloride in step (1)
The synthetic schemes of Resin can avoid using the Rink Amide-AM Resin or Rink Amide-MBHA Resin of high cost
Deng the unfavorable factor of amino resins, effective reduces cost；The raw material for using is 2-chlorotrityl chloride Resin,
Its relative low price, so as to reduce cost on raw material.The 2-chlorotrityl chloride Resin, its name
Referred to as 2- chlorine trityl chloride resin, the present invention uses substitution value for 1.74mmol/g, but other substitution values 2-
Chlorotrityl chloride Resin can also reach same or similar technique effect, also in protection model of the invention
In enclosing.The substitution value of 2-chlorotrityl chloride Resin can be existing commercial all substitution values, the present invention
The substitution value 1.74mmol/g for using is taken from the optimal of the factors such as yield, purity, the utilization rate of resin for synthesizing fragment balance
Value.
Wherein described step (2) is Fmoc-Arg (the pbf) -2-Chlorotrityl Chloride for obtaining step (1)
The de- Fmoc of Resin twice, add DMF, Fmoc-Arg (pbf)-OH, 1- hydroxyls azimidobenzene, the nitrogen of benzo three
Azoles-N, N, N ', N '-tetramethylurea tetrafluoro boric acid, N, N '-diisopropylethylamine react and obtain Fmoc-Arg (pbf)-Arg
(pbf)-2-Chlorotrityl Chloride Resin；Then according to methods described Fmoc-Arg (pbf)-Arg (pbf)-
Fmoc-Gln (Trt)-OH, Fmoc-Met-OH, Fmoc-Glu are sequentially connected on 2-Chlorotrityl Chloride Resin
(OtBu)-OH, Fmoc-Glu (OtBu)-OH, obtain Fmoc-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg
(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin.The step is the step of extending amino acid chain, its
In the protection group of each amino acid side chain protective seletion can be any protection group for having orthogonal protecting effect with Fmoc, as long as energy
The desired technique effect that can be protected enough is reached, but inventor is best by preferred above-mentioned substitution base effect.
Wherein, the step (3) is Fmoc-Glu (OtBu)-Glu (the OtBu)-Met-Gln for obtaining step (2)
(Trt) the de- Fmoc- of-Arg (pbf)-Arg (pbf) -2-Chlorotrityl Chloride Resin twice, add dichloromethane
Alkane, acetic anhydride, N, the mixed liquor of N '-diisopropylethylamine carry out reacting to obtain Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln
(Trt)-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin；Wherein, the step (1), step
(2) reaction is the stirring at normal temperature -2 hours 40 minutes under nitrogen protection and described in step (3)；The step (1), step
(2) take off Fmoc and in step (3) to be with piperidines with the volume ratio of N,N-dimethylformamide is 1:The de- Fmoc of 4 mixed liquor；It is excellent
Select addition dichloromethane, acetic anhydride, N, N '-diisopropylethylamine volume ratio 9 in step (4):1:0.5 mixed liquor；Wherein institute
It is by Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf) -2- to state step (4)
Cutting liquid is added in Chlorotrityl Chloride Resin carries out cleavage reaction by separating, purifying, being dried to obtain Ac-
Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-OH；Wherein step (4) described separation, pure
Change, drying steps are that, by the product filtering after cutting, then filtrate added and cutting with twice saturated common salt water washing 3 times
The isometric saturated aqueous common salt of liquid, pH is adjusted for 7.0,35 DEG C are evaporated off organic solvent and analyse with saturated sodium bicarbonate solution
Go out precipitation, filtering, precipitation uses pure water 3 times, and 40~45 DEG C dry to obtain Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln
(Trt)-Arg(pbf)-Arg(pbf)-OH.Ratio between each material in preparation process can be adjusted suitably, be obtained
Product be still able to meet and require, therefore, to the ratio between each material in preparation process and reaction condition
It is also within the scope of the present invention that the numerical value of parameter carries out appropriate adjustment.In general Solid phase peptide synthssis, if to set
1 is taken on the basis of fat, then the usage amount of amino acid and other reagents will be excessive, can typically take 1.8-3 times, difficult reaction such as occur
The step of dosage may be higher, 1.8-3 times is a dosage for economy.
Wherein described step (5) is by Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg
(pbf)-OH DMFs dissolve, and add hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus, 1-
Hydroxyl azimidobenzene, ammonium chloride, N, N '-diisopropylethylamine react obtaining reaction solution, are processed, separated, purified, dried
Do to obtain Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH2；Then to Ac-Glu
(OtBu) cutting liquid is added to be cut in-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH2, afterwards
Isolated crude product, it is purified, be dried to obtain product.
Wherein, the step (4) cutting liquid is trifluoroethanol and the volume ratio of acetic acid, dichloromethane is 2:0.75:
7.25 mixed liquor；Preferred steps (5) described cutting liquid is that mass percent composition is trifluoroacetic acid 83%, phenol 5%, benzene first
Thioether 4%, water 3%, the mixed liquor of tri isopropyl silane 5%.Above-mentioned each cutting step is different, and the raw material of cutting is different, because
The cutting liquid of this selection is different.
Wherein, 2-chlorotrityl chloride resin and Fmoc-Arg (pbf)-OH, N, N '-two in step (1)
The mol ratio of wopropyl ethyl amine is 1:1:4.
In wherein described the step of (2), 2-chlorotrityl chloride resin and Fmoc-Arg (pbf)-OH,
1- hydroxyls azimidobenzene, BTA-N, N, N ', N '-tetramethylurea tetrafluoro boric acid, N, N '-diisopropylethylamine mole
Than 1:2:(2.5~3):(2.5~3):(2.5~3).In described step (2),
Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-OH and hexafluorophosphoric acid benzene
And triazol-1-yl-epoxide tripyrrole alkyl phosphorus, 1- hydroxyls azimidobenzene, ammonium chloride, N, the mol ratio of N '-diisopropylethylamine
1:3:3:(3~5):(3~5).
Control charge ratio between each material within the above range in preparation process, this is inventor to each step
The result that is obtained by the consideration of Multiple factors of preparation method charge ratio, carried out in above-mentioned protection domain upper and lower 10% it is floating
It is dynamic still to reach described technique effect.Optimal technical scheme is as above.
The present invention also provides the Argireline product that the preparation method is prepared, and its purity is higher than 95%.
In a kind of specific embodiment, the synthetic method of Argireline comprises the following steps：(1) Ac-Glu is synthesized
(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-OH
1. Fmoc-Arg (pbf) -2-Chlorotrityl Chloride Resin are synthesized
By 2-Chlorotrityl Chloride Resin with after swelling 15 minutes of dichloromethane, suction filtration, by 2-
Chlorotrityl Chloride Resin:Fmoc-Arg(pbf)-OH:N, N '-diisopropylethylamine mol ratio are 1:1:4,
It is solvent, under nitrogen protection, stirring at normal temperature that every gram of 2-Chlorotrityl Chloride Resin adds 10mL dichloromethane
1.5 hours, obtain Fmoc-Arg (pbf) -2-Chlorotrityl Chlori de Resin.
2. Fmoc-Arg (pbf)-Arg (pbf) -2-Chlorotrityl Chloride Resin are synthesized
By Fmoc-Arg (pbf) -2-Chlorotrityl Chloride Resin piperidines and N,N-dimethylformamide
Volume ratio be 1:4 mixed liquor takes off Fmoc- twice, by 2-Chlorotrityl Chloride Resin, Fmoc-Arg
(pbf)-OH, 1- hydroxyls azimidobenzene, BTA-N, N, N ', N '-tetramethylurea tetrafluoro boric acid, N, N '-diisopropyl second
Amine mol ratio is 1:2:2.5~3:2.5~3:2.5~3, every gram of 2-Chlorotrityl Chloride Resin is added
10mLN, dinethylformamide is solvent, and under nitrogen protection, stirring at normal temperature 2 hours obtains Fmoc-Arg (pbf)-Arg
(pbf)-2-Chlorotrityl Chlor ide Resin。
3. Fmoc-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf) -2-C is synthesized
hlorotrityl Chloride Resin
According to the method in step (1) 2. to Fmoc-Arg (pbf)-Arg (pbf) -2-Chlorotrityl Chlor
Fmoc-Gln (Trt)-OH, Fmoc-Met-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Glu are sequentially connected on ide Resin
(OtBu)-OH, obtains Fmoc-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf) -2-
Chlorotrityl Chloride Resin。
4. Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf) -2-Chl is synthesized
orotrityl Chloride Resin
By Fmoc-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf) -2-Chlor
Otrityl Chloride Resin piperidines is 1 with the volume ratio of N,N-dimethylformamide:The de- Fmoc- two of 4 mixed liquor
Secondary, by dichloromethane, acetic anhydride, N, N '-diisopropylethylamine volume ratio is 9:1:0.5(v:v:V) reaction solution, every gram of 2- are prepared
Chlorotrityl Chloride Resin add 10mL reaction solutions, and under nitrogen protection, stirring at normal temperature 30 minutes obtains Ac-
Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride
Resin。
5. cutting process
It is 2 by trifluoroethanol, acetic acid, methylene chloride volume ratio:0.75:7.25(v:v:V) cutting liquid, every gram of peptide tree are prepared
Fat Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf) -2-Chlorotrityl
Chloride Resin add 10mL cutting liquids, stirring at normal temperature 1.5 hours, filtering, by filtrate with 2 times of saturated aqueous common salts of volume
Washing 3 times, then adds the saturated aqueous common salt isometric with cutting liquid, and it is 7.0 to adjust PH with saturated sodium bicarbonate solution, rotation
35 DEG C of evaporimeter is evaporated off organic solvent and separates out precipitation, filtering, and precipitation uses pure water 3 times, and 40~45 DEG C dry to obtain Ac-Glu
(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-OH。
(2) Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH2 is synthesized
Every gram of Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-OH is used
10mLN, dinethylformamide dissolving, by Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg
(pbf)-OH, hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus, 1- hydroxyls azimidobenzene, ammonium chloride, N, N '-two
Wopropyl ethyl amine mol ratio is 1:3:3:3~5:3~5 addition hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus,
1- hydroxyls azimidobenzene, ammonium chloride, N, N '-diisopropylethylamine, stirring reaction 3 hours, to the full of reaction solution 2 times of volumes of addition
With saline solution and separate out precipitation, filter, precipitation pure water 3 times, 40~45 DEG C dry to obtain Ac-Glu (OtBu)-Glu
(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-NH2。
(3) synthesis Argireline Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2
It is 83mL in the ratio of trifluoroacetic acid, phenol, thioanisole, water, tri isopropyl silane:5g:4mL:3mL:5mL matches somebody with somebody
Cutting liquid processed, every gram of Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH2 adds 3~
5mL cutting liquids, are stirred at room temperature 1.5~2.5 hours, filtering, by filtrate 5~8 times of cold absolute ethers of addition, separate out precipitation, mistake
Filter, precipitation absolute ether washes 3 times, is vacuum dried, and obtains Argireline crude product, the inverted chromatogram purification of Argireline crude product, lyophilized,
Obtain Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2.
Embodiment
The source of each material to being used in embodiment illustrates below, if not otherwise specified, is used
Raw material and instrument are commercially available, are instrument commonly used in the art and raw material, as long as it can meet experiment needs.
The substitution value of 2-chlorotrityl chloride resin of the invention is 1.74mmol/g, by Tian Jinnan
Open and commercially available into Science and Technology Ltd..
Solid phase peptide synthssis reactor is general Peptide systhesis reactor, can be had purchased from this promise foreign trade of Chengdu
The CS936X CSBio Peptide Synthesizer of limit company.
Analytic type high performance liquid chromatograph is the full-automatic L2000 of Hitachi.
Preparative high performance liquid chromatography instrument is Dalian physio-chemical study for innovation Heng Tong LC3000, C18 analysis chromatographic column
Institute's 4.6mm × 250mm, C18 preparative chromatography post is Chengdu science popularization biology Co., Ltd 40.1mm × 450mm.
Various amino acid used and 1- hydroxyls azimidobenzene, BTA-N, N, N ', N '-tetramethylurea tetrafluoro boric acid,
N, N '-diisopropylethylamine are purchased from Shanghai gill biochemistry Co., Ltd.
LTQ-XL electrospray ionization mass spectrometries instrument is Thermo Finnigan companies of the U.S..
Each organic solvent for being used in experiment, organic reagent are domestic AR or CP.
Embodiment 1
The preparation method of the present embodiment Argireline comprises the following steps：
(1) Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-OH is synthesized
1. Fmoc-Arg (pbf) -2-Chlorotrityl Chloride Resin are synthesized
By in 50.0g2-Chlorotrityl Chloride Resin addition synthesizers, the leaching of 500mL dichloromethane is added
Bubble, after swelling 15 minutes, suction filtration, to added in synthesizer 500mL dichloromethane, 56.45gFmoc-Arg (pbf)-OH,
61.0mLN, N '-diisopropylethylamine, 2-Chlorotrityl Chloride Resin, Fmoc-Arg (pbf)-OH, N, N '-
The mol ratio of diisopropylethylamine is 1:1:4, under nitrogen protection, stirring at normal temperature 1.5 hours, suction filtration, with isopropanol and N, N-
Each washing resin of dimethylformamide 2 times, each 500mL obtains Fmoc-Arg (pbf) -2-Chlorotrityl Chloride
Resin。
2. Fmoc-Arg (pbf)-Arg (pbf) -2-Chlorotrityl Chloride Resin are synthesized
To addition 500mL piperazines in Fmoc-Arg (the pbf) -2-Chlorotrityl Chloride Resin in synthesizer
Pyridine is 1 with the volume ratio of N,N-dimethylformamide:4 mixed liquor reacts 10 minutes, and suction filtration removes mixed liquor, adds
500mL piperidines is 1 with the volume ratio of N,N-dimethylformamide:4 mixed liquor, reacts 20 minutes, suction filtration, with isopropanol and N,
Each washing resin of dinethylformamide 2 times, each 500mL completes Fmoc-Arg (pbf) -2-Chlorotrityl
Chloride Resin take off Fmoc- twice, to addition 500mL DMFs, 112.89g Fmoc- in synthesizer
Arg (pbf)-OH, 35.26g 1- hydroxyls azimidobenzene, 83.81g BTAs-N, N, N ', N '-tetramethylurea tetrafluoro boron
Acid, 45.6mL N, N '-diisopropylethylamine, under nitrogen protection, stirring at normal temperature 2 hours, suction filtration, with isopropanol and N, N- bis-
Each washing resin of NMF 2 times, each 500mL, suction filtration obtains Fmoc-Arg (pbf)-Arg (pbf) -2-
Chlorotrityl Chloride Resin。
3. Fmoc-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf) -2- is synthesized
Chlorotrityl Chloride Resin
According to the method in step (1) 2., to Fmoc-Arg (pbf)-Arg (the pbf) -2- in synthesizer
106.26gFmoc-Gln (Trt)-OH, 64.64gFmoc-Met- are sequentially connected on Chlorotrityl Chloride Resin
OH, 74.04gFmoc-Glu (OtBu)-OH, 74.04gFmoc-Glu (OtBu)-OH, the consumption of other reagents is：1- hydroxy benzenes
Third triazole 35.26g, BTA-N, N, N ', N '-tetramethylurea tetrafluoro boric acid 83.81g, N, N '-diisopropylethylamine
45.6mL, with isopropanol and each washing resin of DMF 2 times, each 500mL obtains Fmoc-Glu (OtBu)-Glu
(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin。
Above-mentioned Fmoc-Gln (Trt)-OH, Fmoc-Met-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Glu (OtBu)-
The mol ratio of OH and 2-chlorotrityl chloride resin is 2:1,2-chlorotrityl chloride
Resin and 1- hydroxyls azimidobenzene, BTA-N, N, N ', N '-tetramethylurea tetrafluoro boric acid, N, N '-diisopropylethylamine
Mol ratio 1:2.5~3:2.5~3:2.5~3.
4. Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf) -2- is synthesized
Chlorotrityl Chloride Resin
To Fmoc-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (the pbf)-Arg (pbf) in synthesizer-
It is 1 with the volume ratio of N,N-dimethylformamide that 500mL piperidines is added in 2-Chlorotrityl Chloride Resin:4
Mixed liquor reacts 10 minutes, and suction filtration removes mixed liquor, and it is 1 to add 500mL piperidines with the volume ratio of DMF:
4 mixed liquor, reacts 20 minutes, suction filtration, with isopropanol and each washing resin of DMF 2 times, each 500mL,
Complete Fmoc-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf) -2-Chlorotrityl
Chloride Resin take off Fmoc- twice, to addition 450mL dichloromethane, 50mL acetic anhydrides, 25mLN, N '-two in synthesizer
Wopropyl ethyl amine, under nitrogen protection, stirring at normal temperature 30 minutes, suction filtration is respectively washed with isopropanol and DMF
Resin 2 times, each 500mL, suction filtration, methyl alcohol washing resin 3 times, each 500mL, suction filtration is dried, and obtains Ac-Glu (OtBu)-Glu
(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin。
5. cutting process
To Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf) -2-
Chlorotrityl Chloride Resin add 500mL cutting liquids, and cutting liquid used is trifluoroethanol, acetic acid, dichloromethane
Alkane volume ratio is 2:0.75:7.25(v:v:Filtrate is moved into 1000mL points by mixed liquor v), stirring at normal temperature 1.5 hours, filtering
In liquid funnel, with 1000mL saturated common salts water washing 3 times, then filtrate moved into 2000mL eggplants type bottle, add 500mL saturations food
Salt solution, it is 7.0 to adjust PH with saturated sodium bicarbonate solution, and 35 DEG C of Rotary Evaporators are evaporated off organic solvent and separate out precipitation, with No. 4 sand
Core funnel suction filtration, precipitation pure water 3 times, 40~45 DEG C of drying, obtain 120.16gAc-Glu (OtBu)-Glu (OtBu)-
Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-OH, yield 78.94%.
(2) Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH is synthesized2
By 120.16g (68.68mmol) Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg
(pbf) in-OH additions 5L beakers, addition 1200mLN, dinethylformamide, 107.22g hexafluorophosphoric acid BTA -1- bases -
Epoxide tripyrrole alkyl phosphorus, 27.84g1- hydroxyls azimidobenzene, 18.37g ammonium chlorides, 59.8mLN, N '-diisopropylethylamine, stir
Reaction 3 hours is mixed, is added to reaction solution and 2400mL saturated aqueous common salts and is separated out precipitation, with No. 4 sand core funnel suction filtrations, precipitated with pure
Water washing 3 times, 40~45 DEG C drying, obtain 113.11gAc-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-
Arg (pbf)-NH2, yield 94.13%.
(3) synthesis Argireline Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2
To 113.11gAc-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-NH2In
Add 300mL cutting liquids, cutting liquid used is that trifluoroacetic acid, phenol, thioanisole, water, tri isopropyl silane ratio are
83mL:5g:4mL:3mL:5mL mixed solutions, are stirred at room temperature 2 hours, with No. 3 sand core funnel suction filtrations, filtrate are added into 2400mL
In cold absolute ether, precipitation is separated out, with No. 4 sand core funnel suction filtrations, precipitation absolute ether is washed 3 times, vacuum drying obtains six victory
Peptide crude product, it is the inverted chromatogram purification of Argireline crude product, lyophilized, obtain 63.35g Argirelines, yield 81.79%.
The structure of synthetic product is characterized using mass spectrograph, as a result sees Fig. 1, as seen from the figure, synthesized product
Molecular weight and molecular ion peak are consistent with the molecular weight and molecular ion peak of Argireline, illustrate that synthetic product is Argireline.Using
Liquid chromatograph carries out purity testing to the Argireline for synthesizing, and liquid chromatogram is shown in Fig. 2 (a), visible with reference to Fig. 2 (b), Argireline
Purity be more than 95%.
Embodiment 2
In the step of this implementation (1), Fmoc-Arg (pbf)-OH, Fmoc-Gln (Trt)-OH, Fmoc-Met-OH, Fmoc-
Glu (OtBu)-OH, Fmoc-Glu (OtBu)-OH are 2 with the mol ratio of 2-Chlorotrityl Chloride Resin:1,
2-Chlorotrityl Chloride Resin and 1- hydroxyls azimidobenzene, BTA-N, N, N ', N '-tetramethylurea four
Fluoboric acid, N, the mol ratio of N '-diisopropylethylamine is 1:2.5:2.5:2.5, other steps and embodiment (1) phase of the step
Together.In the step of the present embodiment (2), Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-
OH, hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus, 1- hydroxyls azimidobenzene, ammonium chloride, N, N '-diisopropyl
Ethamine mol ratio is 1:3:3:3:3, other steps of the step are identical with embodiment (1).The step of this implementation (3) and embodiment
(1) it is identical, obtain 62.51g Argirelines, yield 80.71%.Mass spectrograph pairing is used using the identical condition on production of embodiment 1
Structure into product is characterized, the molecular weight and molecular ion peak of synthesized product and the molecular weight and molecule of Argireline from
Sub- peak is consistent.Purity testing is carried out to the Argireline for synthesizing using liquid chromatograph, purity is more than 95%.
Embodiment 3
The step of this implementation (1), is identical with embodiment (1).In the step of the present embodiment (2), Ac-Glu (OtBu)-Glu
(OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-OH, hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl
Phosphorus, 1- hydroxyls azimidobenzene, ammonium chloride, N, N '-diisopropylethylamine mol ratio are 1:3:3:4:4, other steps of the step
It is identical with embodiment (1).The step of this implementation (3), is identical with embodiment (1), obtains 62.78g Argirelines, yield 81.06%.Adopt
The structure of synthetic product is characterized using mass spectrograph with the identical condition on production of embodiment 1, synthesized product point
Son amount and molecular ion peak are consistent with the molecular weight and molecular ion peak of Argireline.Using liquid chromatograph to synthesize Argireline
Purity testing is carried out, purity is more than 95%.
Claims (14)
1. a kind of preparation method of Argireline, it is characterised in that methods described comprises the following steps：
(1) synthesis Fmoc-Arg (pbf) -2-Chlorotrityl Chloride Resin；
(2) Fmoc-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf) -2- is synthesized
Chlorotrityl Chloride Resin；
(3) Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf) -2- is synthesized
Chlorotrityl Chloride Resin；
(4) Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-OH is synthesized；And
(5) step (4) is obtained into product to be cut, separate and purifying obtains Argireline product.
2. the preparation method of Argireline as claimed in claim 1, wherein the step (1) is by 2-Chlorotrityl
Chloride Resin with dichloromethane it is swelling after, add dichloromethane, Fmoc-Arg (pbf)-OH, N, N '-diisopropyl second
Amine react and obtains Fmoc-Arg (pbf) -2-Chlorotrityl Chloride Resin.
3. the preparation method of Argireline as claimed in claim 1 or 2, wherein the step (2) obtains step (1)
Fmoc-Arg (pbf) -2-Chlorotrityl Chloride Resin take off Fmoc twice, addition DMF,
Fmoc-Arg (pbf)-OH, 1- hydroxyls azimidobenzene, BTA-N, N, N ', N '-tetramethylurea tetrafluoro boric acid, N, N '-two
Wopropyl ethyl amine react and obtains Fmoc-Arg (pbf)-Arg (pbf) -2-Chlorotrityl Chloride Resin；So
Connect successively on Fmoc-Arg (pbf)-Arg (pbf) -2-Chlorotrityl Chloride Resin according to methods described afterwards
Fmoc-Gln (Trt)-OH, Fmoc-Met-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Glu (OtBu)-OH are met, Fmoc- is obtained
Glu(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride
Resin。
4. as described in claim any one of 1-3 Argireline preparation method, wherein the step (3) is to obtain step (2)
Fmoc-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf) -2-Chlorotrityl
The de- Fmoc- of Chloride Resin twice, add dichloromethane, acetic anhydride, N, the mixed liquor of N '-diisopropylethylamine to carry out instead
Deserved Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf) -2-Chlorotrityl
Chloride Resin。
5. reaction is protected in nitrogen described in step (1), step (2) and step (3) as described in claim any one of 2-4
Under, stirring at normal temperature 0.5-2 hours；It is with piperidines and N, N- dimethyl that Fmoc is taken off in the step (1), step (2) and step (3)
The volume ratio of formamide is 1:The de- Fmoc of 4 mixed liquor；Dichloromethane, acetic anhydride, N are added in preferred steps (4), N '-two is different
Propylethylamine volume ratio 9:1:0.5 mixed liquor.
6. as described in claim any one of 1-5 Argireline preparation method, wherein the step (4) is by Ac-Glu
(OtBu)-Glu(OtBu)-Met-Gln(Trt)-Arg(pbf)-Arg(pbf)-2-Chlorotrityl Chloride Resin
Middle addition cutting liquid carries out cleavage reaction by separating, purifying, being dried to obtain Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln
(Trt)-Arg(pbf)-Arg(pbf)-OH。
7. the preparation method of Argireline as claimed in claim 6, wherein step (4) described separation, purifying, drying steps are to cut
Then product filtering after cutting, filtrate add the saturated common salt isometric with cutting liquid with twice saturated common salt water washing 3 times
Water, adjusts pH for 7.0,35 DEG C are evaporated off organic solvent and separate out precipitation with saturated sodium bicarbonate solution, filters, and precipitation is used
Pure water 3 times, 40~45 DEG C dry to obtain Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg
(pbf)-OH。
8. as described in claim any one of 1-7 Argireline preparation method, wherein the step (5) is by Ac-Glu
(OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-OH DMFs dissolve, and add
Hexafluorophosphoric acid BTA -1- bases-epoxide tripyrrole alkyl phosphorus, 1- hydroxyls azimidobenzene, ammonium chloride, N, N '-diisopropyl second
Amine react obtaining reaction solution, is processed, separated, purified, dried and to obtain Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln
(Trt)-Arg(pbf)-Arg(pbf)-NH2；Then to Ac-Glu (OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg
(pbf) cutting liquid is added to be cut in-Arg (pbf)-NH2, isolated crude product afterwards is purified, be dried to obtain product.
9. the preparation method of Argireline as claimed in claim 8, wherein through filtering after the step (5) cutting, by filtrate plus
Enter in cold diethyl ether, separate out precipitation, obtain Argireline crude product, the inverted chromatogram purification of Argireline crude product, freeze-drying obtain Ac-
Glu-Glu-Met-Gln-Arg-Arg-NH2 products.
10. as described in claim any one of 6-8 Argireline preparation method, wherein the step (4) described cutting liquid is three
Fluoroethanol is 2 with the volume ratio of acetic acid, dichloromethane:0.75:7.25 mixed liquor；Preferred steps (5) described cutting liquid is matter
Amount percentage composition is trifluoroacetic acid 83%, phenol 5%, thioanisole 4%, water 3%, the mixed liquor of tri isopropyl silane 5%.
11. as described in claim any one of 2-10 Argireline preparation method, wherein 2-chlorotrityl in step (1)
Chloride resin and Fmoc-Arg (pbf)-OH, N, the mol ratio of N '-diisopropylethylamine is 1:1:4.
The synthetic method of 12. Argireline according to claim any one of 2-11, it is characterised in that：Described step (2)
In, 2-chlorotrityl chloride resin and Fmoc-Arg (pbf)-OH, 1- hydroxyls azimidobenzene, BTA-
N, N, N ', N '-tetramethylurea tetrafluoro boric acid, N, the mol ratio 1 of N '-diisopropylethylamine:2:(2.5~3):(2.5~3):
(2.5~3).
The synthetic method of 13. Argirelines according to claim 1, it is characterised in that：In described step (2), Ac-Glu
(OtBu)-Glu (OtBu)-Met-Gln (Trt)-Arg (pbf)-Arg (pbf)-OH and hexafluorophosphoric acid BTA -1- bases-oxygen
Base tripyrrole alkyl phosphorus, 1- hydroxyls azimidobenzene, ammonium chloride, N, the mol ratio 1 of N '-diisopropylethylamine:3:3:(3~5):(3
~5).
The Argireline product that preparation method described in 14. claim any one of 1-13 is prepared, its purity is higher than 95%.
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|---|---|---|---|---|
| CN110684077A (en) * | 2019-10-18 | 2020-01-14 | 陕西慧康生物科技有限责任公司 | Large-scale synthesis method of achirelin |
| CN116987148A (en) * | 2023-08-10 | 2023-11-03 | 山东济肽生物科技有限公司 | Synthesis process of acetyl hexapeptide-8 |
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| CN114805476A (en) * | 2022-04-14 | 2022-07-29 | 南京莱昂生物科技有限公司 | Preparation method of cetyl ester |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102603869A (en) * | 2012-03-27 | 2012-07-25 | 西安华澳丽康生物工程有限公司 | Synthetic method of hexapeptide |
| CN103694316A (en) * | 2013-12-31 | 2014-04-02 | 杭州华津药业股份有限公司 | Preparation method of argireline |
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| CN103613642B (en) * | 2013-11-20 | 2015-05-13 | 陕西东大生化科技有限责任公司 | Liquid-phase segmented synthesis method of argireline |
-
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102603869A (en) * | 2012-03-27 | 2012-07-25 | 西安华澳丽康生物工程有限公司 | Synthetic method of hexapeptide |
| CN103694316A (en) * | 2013-12-31 | 2014-04-02 | 杭州华津药业股份有限公司 | Preparation method of argireline |
Non-Patent Citations (2)
| Title |
|---|
| 宋乃建等: "六胜肽的固相合成", 《化学工程师》 * |
| 王莹等: "多肽的固相合成法研究进展", 《安徽农业科学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110684077A (en) * | 2019-10-18 | 2020-01-14 | 陕西慧康生物科技有限责任公司 | Large-scale synthesis method of achirelin |
| CN116987148A (en) * | 2023-08-10 | 2023-11-03 | 山东济肽生物科技有限公司 | Synthesis process of acetyl hexapeptide-8 |
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| WO2018126525A1 (en) | 2018-07-12 |
| AU2017101705A4 (en) | 2018-03-01 |
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