CN109734778A - A kind of preparation method of Wella card peptide - Google Patents
A kind of preparation method of Wella card peptide Download PDFInfo
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- CN109734778A CN109734778A CN201910172421.5A CN201910172421A CN109734778A CN 109734778 A CN109734778 A CN 109734778A CN 201910172421 A CN201910172421 A CN 201910172421A CN 109734778 A CN109734778 A CN 109734778A
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Abstract
The present invention provides a kind of preparation methods of Wella card peptide, belong to polypeptide drugs preparation technical field.The present invention is raw material using Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-D-Arg (Pbf)-OH, D-Arg residue is accessed in the form of D-Arg-D-Arg-D-Arg tripeptide fragment, efficiently avoids the generation of peptide disappearance impurity [- 1-D-Arg]-Wella card peptide, [- 2-D-Arg]-Wella card peptide and [- 3-D-Arg]-Wella card peptide；Simultaneously, the present invention carries out coupling reaction with Boc-L-Cys-OH again after activating the Cys side chain thiol of de- Mmt Wella card peptide-amino main chain peptide resin, the disulfide bond of Wella card peptide can be successfully constructed, and preparation method combined coefficient provided by the invention is high, be suitble to industrialization large-scale production.
Description
Technical field
The present invention relates to polypeptide drugs preparation technical field, in particular to a kind of preparation method of Wella card peptide.
Background technique
Secondary hyperparathyroidism (SHPT, abbreviation Secondary Hyperparathyroidism), refers in chronic renal insufficiency, intestines
Malabsorption syndrome, Fanconi syndrome and the feelings such as renal tubular acidosis, vitamin D deficiency or resistance and gestation, lactation
Under condition, parathyroid gland for a long time by low blood calcium, hypomagnesemia or hyperphospheremia stimulation and secrete excessive parathyroid hormone
(PTH), to improve blood calcium, blood magnesium and a kind of chronic compensatory clinical manifestation for reducing serium inorganic phosphorus, long-term parathyroid hyperplasia is most
The autonomous adenoma of function is resulted in eventually.
Wella card peptide is by KaiPharmaceuticals, a kind of novel Sensipar of Inc. exploitation
(calcimimeticagent), it is able to suppress the secretion of parathyroid hormone.Wella card peptide is combinable and activates on parathyroid gland
Calcium-sensing receptor, realize the reduction of parathyroid hormone level.
Wella card peptide has arginine amide, the 1 L structure of the arginine of 3 D configurations, the alanine of 2 D configurations, 1 D configuration
Type cysteine and 1 D configuration cysteine (N sections are closed by acetyl group) are constituted, wherein D configuration cysteine and L-configuration half
Cystine is linked together (N-acetyl-D-cysteinyl-D-alanyl-D-arginyl-D-arginyl-D- with disulfide bond
Arginyl-D-alanyl-D-Argininamide, disulfidewithL-cysteine), shown in structural formula I:
The key of the compound synthesis is the building of disulfide bond, and the side of disulfide bond is constructed used in conventional peptide synthesis
Method includes air oxidation process, iodine/acetate system oxidizing process, hydrogen peroxide oxidation method etc., and such methods are appropriate only for two sulphur of cyclic peptide
The building of key, but Wella card peptide needs to carry out the oxidation reaction of intermolecular sulfydryl, if according to conventional disulfide bond construction method
It is reacted, will be unable to obtain the final product of ideal recovery and purity.
Meanwhile arginine (Arg) belongs to difficult amino acid, the very easy peptide disappearance for generating [- D-Arg], and Wella card
Peptide sequence contains 3 continuous D-Arg residues in, easily generation peptide disappearance impurity [- 1-D-Arg]-Wella card peptide, [- 2-D-
Arg]-Wella card peptide and [- 3-D-Arg]-Wella card peptide.These impurity are close with the polarity of target peptide, are not readily separated, and increase and produce
The difficulty that product isolate and purify.
Therefore, it needs to solve the building difficulty of disulfide bond in the preparation process of Wella card peptide and is also easy to produce asking for peptide disappearance
Topic.
Summary of the invention
In view of this, it is an object of that present invention to provide a kind of preparation methods of Wella card peptide.Wella card provided by the invention
The problem of preparation method of peptide is able to solve the building difficulty of disulfide bond and is also easy to produce peptide disappearance, the life suitable for industrial mass
It produces, and the yield of gained Wella card peptide is high, purity is high.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of preparation methods of Wella card peptide, comprising the following steps:
(1) under condensation system effect, using fmoc-protected amino resins as carrier, by full guard amino acid and Fmoc-
D-Arg (Pbf)-D-Arg (Pbf)-D-Arg (Pbf)-OH tripeptide fragment is condensed, and the dimension with structure shown in formula (a) is obtained
Draw card peptide-amino main chain peptide resin；Sequence of the condensation according to Wella card peptide backbone peptide from C-terminal to N-terminal carries out；
The full guard amino acid and Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-D-Arg (Pbf)-OH tripeptide fragment
Condensation sequence are as follows: Fmoc-D-Arg (Pbf)-OH, Fmoc-D-Ala-OH, Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-D-Arg
(Pbf)-OH,Fmoc-D-Ala-OH,N-Ac-D-Cys(Mmt)-OH；
(2) by the Wella card peptide-amino main chain peptide resin and Mmt protection solution is gone to mix, it is anti-carries out de- Mmt protection
It answers, obtains the de- Mmt Wella card peptide-amino main chain peptide resin with structure shown in formula (b)；
(3) after the Cys side chain thiol of the de- Mmt Wella card peptide-amino main chain peptide resin being activated with Boc-L-Cys-
OH mixing, carries out coupling reaction, obtains the full guard Wella card peptide-amino resins with structure shown in formula (c)；
(4) the full guard Wella card peptide-amino resins and lytic reagent are mixed and carries out cracking reaction, obtain Wella card
Peptide.
Preferably, Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-D-Arg (the Pbf)-OH tripeptide fragment is by following methods
It is prepared:
(I) Fmoc-D-Arg (Pbf)-OH and HOSu is subjected under the action of DCC coupling reaction, obtains Fmoc-D-Arg
(Pbf)-OSu；
(II) Fmoc-D-Arg (Pbf)-OSu and H-D-Arg (Pbf)-OH are carried out under the action of base catalyst
Coupling reaction obtains Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-OH；
(III) Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-OH and HOSu is carried out being coupled under the action of DCC anti-
It answers, obtains Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-OSu；
(IV) by the Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-OSu and H-D-Arg (Pbf)-OH in base catalyst
Effect is lower to carry out coupling reaction, obtains Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-D-Arg (Pbf)-OH.
Preferably, the base catalyst in the step (II) and step (IV) is Na2CO3。
Preferably, the step (1) specifically:
I) by after the swelling of Fmoc amino resins, de- Fmoc protection is carried out in the case where going Fmoc to protect solution effects, obtains remove-insurance
The amino resins of shield；
Ii) by the full guard amino acid, Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-D-Arg (Pbf)-OH tripeptides piece
Section is mixed with condensation system, carries out priming reaction, the amino acid and tripeptide fragment activated；
Iii the amino acid of the activation and tripeptide fragment are mixed with the amino resins of the deprotection), be condensed anti-
It answers, obtains Wella card peptide-amino main chain peptide resin；
The step i) and step ii) not no time sequencing limitation.
Preferably, the condensation system includes condensing agent and reaction dissolvent, and the condensing agent is DIC/HOBt, PyBOP/
HOBt/DIEA or HATU/HOBt/DIEA；The reaction dissolvent is one or more of DMF, DCM, NMP and DMSO；
The molar ratio of the full guard amino acid, condensing agent and Fmoc amino resins is 2~5:2~5:1.
Preferably, the amino resins is RinkAmide resin, RinkAmideMBHA resin and RinkAmideAM resin
One or more of；The degree of substitution of the amino resins is 0.2~1.0mmol/g.
Preferably, swelling with sweller is DCM or DMF in the step i)；It is described go Fmoc protection solution be piperidines and
The mixed liquor of DMF.
Preferably, go Mmt protection solution for the DCM solution of TFA in the step (2), it is described to go in Mmt protection solution
The volume content of TFA is 1~3%.
Preferably, the activation side of the Cys side chain thiol of Mmt Wella card peptide-amino main chain peptide resin is taken off in the step (3)
Method are as follows:
The de- Mmt Wella card peptide-amino main chain peptide resin is mixed with activator, priming reaction is carried out, obtains the side Cys
The sulfhydryl activated Wella card peptide-amino main chain peptide resin of chain；The activator is bis- sulphur of 2,2'-, two pyridine.
Preferably, the lytic reagent in the step (4) is TFA, thioanisole, TIS, EDT and H2The mixed solvent of O,
The in the mixed solvent TFA, thioanisole, TIS, EDT and H2The volume ratio of O is 80~90:10~5:5~2:5~2:1.
The present invention provides a kind of preparation method of Wella card peptide, the present invention uses Fmoc-D-Arg (Pbf)-D-Arg
(Pbf)-D-Arg (Pbf)-OH is raw material, and D-Arg residue is accessed in the form of D-Arg-D-Arg-D-Arg tripeptide fragment, is had
Avoid peptide disappearance impurity [- 1-D-Arg]-Wella card peptide, [- 2-D-Arg]-Wella card peptide and [- 3-D-Arg]-Wella to effect
The generation of card peptide；Meanwhile the present invention Cys side chain thiol of de- Mmt Wella card peptide-amino main chain peptide resin is activated after again with
Boc-L-Cys-OH carries out coupling reaction, can successfully construct the disulfide bond of Wella card peptide, and preparation side provided by the invention
Method combined coefficient is high, is suitble to industrialization large-scale production.Embodiment the result shows that, use Wella obtained by preparation method of the invention
The yield of card peptide is up to 79.3%, and purity is up to 98.50%.
Detailed description of the invention
Fig. 1 is the mass spectrogram of 6 gained Wella card peptide of the embodiment of the present invention.
Specific embodiment
The concrete meaning of abbreviation used herein is shown in Table 1:
The concrete meaning that table 1 is respectively abridged
The present invention provides a kind of preparation methods of Wella card peptide, comprising the following steps:
(1) under condensation system effect, using fmoc-protected amino resins as carrier, by full guard amino acid and Fmoc-
D-Arg (Pbf)-D-Arg (Pbf)-D-Arg (Pbf)-OH tripeptide fragment is condensed, and the Wella card with formula (a) structure is obtained
Peptide-amino main chain peptide resin；Sequence of the condensation according to Wella card peptide backbone peptide from C-terminal to N-terminal carries out；
The full guard amino acid and Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-D-Arg (Pbf)-OH tripeptide fragment
Condensation sequence are as follows: Fmoc-D-Arg (Pbf)-OH, Fmoc-D-Ala-OH, Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-D-Arg
(Pbf)-OH,Fmoc-D-Ala-OH,N-Ac-D-Cys(Mmt)-OH；
(2) by the Wella card peptide-amino main chain peptide resin and Mmt protection solution is gone to mix, it is anti-carries out de- Mmt protection
It answers, obtains the de- Mmt Wella card peptide-amino main chain peptide resin with formula (b) structure；
(3) after the Cys side chain thiol of the de- Mmt Wella card peptide-amino main chain peptide resin being activated with Boc-L-Cys-
OH mixing, carries out coupling reaction, obtains the full guard Wella card peptide-amino resins with structure shown in formula (c)；
(4) the full guard Wella card peptide-amino resins and lytic reagent are mixed and carries out cracking reaction, obtain Wella card
Peptide.
In the present invention, Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-D-Arg (the Pbf)-OH tripeptide fragment preferably by
Following methods are prepared:
(I) Fmoc-D-Arg (Pbf)-OH and HOSu is subjected under the action of DCC coupling reaction, obtains Fmoc-D-Arg
(Pbf)-OSu；
(II) Fmoc-D-Arg (Pbf)-OSu and H-D-Arg (Pbf)-OH are carried out under the action of base catalyst
Coupling reaction obtains Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-OH；
(III) Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-OH and HOSu is carried out being coupled under the action of DCC anti-
It answers, obtains Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-OSu；
(IV) by the Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-OSu, H-D-Arg (Pbf)-OH base catalysis work
Coupling reaction is carried out under, obtains Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-D-Arg (Pbf)-OH.
In the present invention, the molar ratio of Fmoc-D-Arg (Pbf)-OH, HOSu and DCC are preferably 1 in the step (I):
1.0~1.5:1.0~1.5, more preferably 1:1.1:1.1.
In the present invention, the solvent of coupling reaction is preferably THF, use of the present invention to the solvent in the step (I)
No particular/special requirement is measured, reaction can be made to go on smoothly；In the present invention, the coupling reaction in the step (I) is preferred
Including the low-temp reaction stage and room temperature reaction stage successively carried out, the temperature in the low-temp reaction stage is preferably 0 DEG C, the time
Preferably 0.5~2h, more preferably 1h；The temperature in the room temperature reaction stage is preferably 25 DEG C, and the time is preferably 3h；In this hair
In bright, the DCC is the condensation reagent of coupling reaction.
In a specific embodiment of the present invention, preferably first Fmoc-D-Arg (Pbf)-OH, HOSu are dissolved in solvent, are obtained
Mixed liquor；DCC is dissolved in solvent, DCC solution is obtained；Then DCC solution is added dropwise into gained mixed liquor, in ice-water bath condition
Lower carry out low-temp reaction after the completion of low-temp reaction, reacting liquid temperature is warmed to room temperature, starts to be reacted at room temperature.In the present invention
In, the time of the low-temp reaction is added dropwise with the DCC solution to be started to calculate；The time of the room temperature reaction is warming up to certainly
Start to calculate when the temperature in room temperature reaction stage.
After the completion of the coupling reaction of step (I), the present invention preferably post-processes coupling reaction solution, and the post-processing is excellent
Choosing is evaporated the following steps are included: being successively filtered coupling reaction liquid with first, obtains being evaporated object；Object dissolution is evaporated by described
It is successively filtered after in DCM and is evaporated with second, obtain crude product；The use of ethyl acetate is solvent, the crude product is carried out
Recrystallization, obtains Fmoc-D-Arg (Pbf)-OSu.The present invention is to the filtering, the concrete mode for being evaporated and recrystallizing without spy
Different requirement using filtering well known to those skilled in the art, is evaporated and recrystallization operation.
After obtaining Fmoc-D-Arg (Pbf)-OSu, the present invention is preferably by the Fmoc-D-Arg (Pbf)-OSu, H-D-Arg
(Pbf)-OH carries out coupling reaction under the action of base catalyst, obtains Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-OH.At this
In invention, the molar ratio of Fmoc-D-Arg (the Pbf)-OSu, H-D-Arg (Pbf)-OH and base catalyst is preferably 1:1.2:
1.2, the base catalyst is preferably Na2CO3；In the present invention, the temperature of coupling reaction is preferably 25 in the step (II)
DEG C, the time is preferably 8~12h；The solvent of coupling reaction is preferably THF-H in the step (II)2O mixed solvent, the present invention
There is no particular/special requirement to the dosage of the solvent, reaction can be made to go on smoothly.
In a specific embodiment of the present invention, in the step (II) the step of coupling reaction specifically:
H-D-Arg (Pbf)-OH, base catalyst are dissolved in THF aqueous solution, mixed liquor is obtained；
Fmoc-D-Arg (Pbf)-OSu is dissolved in THF, Fmoc-D-Arg (Pbf)-OSu solution is obtained；
Fmoc-D-Arg (Pbf)-OSu solution is added dropwise into the mixed liquor, carries out coupling reaction.
In the present invention, the volume fraction of THF in the THF aqueous solution of H-D-Arg (Pbf)-OH and base catalyst is dissolved
Preferably 50%；Dissolve THF aqueous solution and dissolution Fmoc-D-Arg (Pbf)-of H-D-Arg (Pbf)-OH and base catalyst
The THF of OSu collectively forms the dicyandiamide solution of coupling reaction in step (II)；The time being coupled in the step (II) is certainly
Fmoc-D-Arg (Pbf)-OSu solution starts to calculate after being added dropwise to complete.
In step (II) after the completion of coupling reaction, the present invention preferably post-processes gained coupling reaction liquid, after described
Processing preferably includes following steps: the coupling reaction liquid successively being rotated, extracts, washs, dries and recrystallized, is obtained
Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-OH solid.
In the present invention, the temperature of the revolving is preferably 30~35 DEG C, and the time is preferably 2~5h.The present invention passes through rotation
It boils off except the solvent in coupling reaction liquid.In the present invention, the extraction is preferably ethyl acetate with extractant, and the present invention is preferred
The pH value for rotating liquid is adjusted to 3 using the citric acid that volume fraction is 15% before extraction；In the present invention, the washing is with washing
It washs agent and is preferably saturated NaCl solution；The drying is preferably anhydrous Na with desiccant2SO4.The recrystallization is preferably with solvent
Ethyl acetate.
After obtaining Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-OH, the present invention is preferably by Fmoc-D-Arg (Pbf)-D-Arg
(Pbf)-OH, HOSu are mixed with DCC, are carried out coupling reaction, are obtained Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-OSu.In this hair
In bright, the molar ratio of Fmoc-D-Arg (Pbf)-D-Arg (the Pbf)-OH, HOSu and DCC are preferably 1:1.1:1.1；It is described
The mode of operation of coupling reaction and post processing mode are identical with the step (I) in step (III), only replace raw material
It changes, details are not described herein.
After obtaining Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-OSu, the present invention is preferably by the Fmoc-D-Arg (Pbf)-
D-Arg (Pbf)-OSu, H-D-Arg (Pbf)-OH and base catalyst mixing, carry out coupling reaction, obtain Fmoc-D-Arg
(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-OH.In the present invention, Fmoc-D-Arg (Pbf)-D-Arg (the Pbf)-OSu,
The molar ratio of H-D-Arg (Pbf)-OH and base catalyst is preferably 1:1.2:1.2, and the base catalyst is preferably Na2CO3.At this
In invention, the mode of operation of coupling reaction and post processing mode are identical as the step (II) in the step (IV), only will
Raw material is replaced, and details are not described herein.
After obtaining Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-D-Arg (Pbf)-OH, the present invention is in condensation system effect
Under, using fmoc-protected amino resins as carrier, by full guard amino acid and Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-D-
Arg (Pbf)-OH tripeptide fragment is condensed, and the Wella card peptide-amino main chain peptide resin with formula (a) structure is obtained.In this hair
In bright, the condensation is particularly preferred as:
I) by after the swelling of Fmoc amino resins, de- Fmoc protection is carried out in the case where going Fmoc to protect solution effects, obtains remove-insurance
The amino resins of shield；
Ii) by the full guard amino acid, Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-D-Arg (Pbf)-OH tripeptides piece
Section is mixed with condensation system, carries out priming reaction, the tripeptide fragment of the amino acid and activation that are activated；
Iii the amino acid of the activation and tripeptide fragment are mixed with the amino resins of the deprotection), be condensed anti-
It answers, obtains Wella card peptide-amino main chain peptide resin；
The step i) and step ii) not no time sequencing limitation.
After Fmoc amino resins is swollen by the present invention, de- Fmoc protection is carried out in the case where going Fmoc to protect solution effects, is obtained
The amino resins of deprotection.In the present invention, the amino resins is preferably RinkAmide resin, RinkAmideMBHA resin
One or more of with RinkAmideAM resin；The degree of substitution of the amino resins is preferably 0.2~1.0mmol/g, more excellent
It is selected as 0.4~0.8mmol/g.In the present invention, the swelling is preferably DCM and/or DMF with sweller；The sweller body
The long-pending mass ratio with Fmoc amino resins is preferably 10mL:1g, and the time of the swelling is preferably 0.5h.The present invention is preferably molten
The Fmoc amino resins is washed after swollen, the washing is preferably DMF with detergent.
In the present invention, it is described go Fmoc protection solution be preferably piperidines and the mixed liquor of DMF；Piperidines in the mixed liquor
Volume ratio with DMF is preferably 1:4.In the present invention, it is de- to preferably include the first time successively carried out for the de- Fmoc protection
Fmoc protection and second of de- Fmoc protection, specifically: deprotection solution is added in swelling amino resins solid, is taken off
Fmoc protection obtains the first de- Fmoc protection amino resins；Deprotection solution is added to the described first de- Fmoc again and protects ammonia
In base resin, second of de- Fmoc protection is carried out, de- Fmoc protection amino resins is obtained.In the present invention, the first time is de-
Fmoc-protected temperature is preferably 15~35 DEG C, and the time is preferably 5min；It is described to take off fmoc-protected temperature for the second time and be preferably
15~35 DEG C, the time is preferably 10min.The present invention is using de- Fmoc protection twice, it is ensured that the completely de- Fmoc of amino resins
Protection.
After completing de- Fmoc protection, the present invention preferably washs the amino resins of the deprotection to neutrality, the washing
It is preferably DMF with detergent.
The present invention is preferably by the full guard amino acid, Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-D-Arg (Pbf)-OH
Tripeptide fragment is mixed with condensation system, carries out priming reaction, the tripeptide fragment of the amino acid and activation that are activated.In the present invention
In, the condensation system preferably includes condensing agent and reaction dissolvent, and the condensing agent is preferably DIC/HOBt, PyBOP/HOBt/
DIEA or HATU/HOBt/DIEA；The reaction dissolvent is preferably one or more of DMF, DCM, NMP and DMSO.In this hair
In bright, the temperature of the priming reaction is preferably 15~30 DEG C, and more preferably 20~25 DEG C；The time of the priming reaction is preferred
For 5~10min, more preferably 6~8min.
After the amino acid of amino resins and activation and the tripeptide fragment of activation that are deprotected, the present invention preferably will be described
The amino acid of activation and the tripeptide fragment of activation are mixed with the amino resins of the deprotection, are carried out condensation reaction, are obtained Wella
Card peptide-amino main chain peptide resin.In the present invention, the sequence of the condensation according to Wella card peptide backbone peptide from C-terminal to N-terminal into
Row；The condensation of the full guard amino acid and Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-D-Arg (Pbf)-OH tripeptide fragment is suitable
Sequence are as follows: Fmoc-D-Arg (Pbf)-OH, Fmoc-D-Ala-OH, Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-D-Arg (Pbf)-
OH,Fmoc-D-Ala-OH,N-Ac-D-Cys(Mmt)-OH.In the present invention, the temperature of the condensation reaction is preferably 15~30
DEG C, more preferably 20~25 DEG C；The time of the condensation reaction is preferably 1~5h, more preferably 3~4h.In the present invention, institute
The molar ratio for stating full guard amino acid, condensing agent and Fmoc amino resins is preferably 2~5:2~5:1, and more preferably 3~4:3~
4:1.
Present invention preferably uses the methods of ninhydrin reaction colour developing to monitor condensation reaction process, the ninhydrin reaction colour developing
Method specifically: take a small amount of condensation reaction products as in test tube, washed twice using DMF, in test tube be added one drop body
After 5% ninhydrin of fraction-ethanol solution, 85% phenol of drop volume score-ethanol solution and a drop pyridine, in 120 DEG C of items
2min is heated under part, then after being washed twice with DMF, observe condensation reaction products color, if resin is yellow, illustrate this amino
Acid or peptide fragment condensation completely, can carry out the condensation of next amino acid or peptide fragment；If resin is blue, illustrate this amino
Acid or peptide fragment are not condensed completely, need to be condensed again.
The present invention is raw material using Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-D-Arg (Pbf)-OH, by D-Arg residue
It is accessed in the form of D-Arg-D-Arg-D-Arg tripeptide fragment, effectively avoids peptide disappearance impurity [- 1-D-Arg]-Wella card
The generation of peptide, [- 2-D-Arg]-Wella card peptide and [- 3-D-Arg]-Wella card peptide.
In the present invention, the preparation of Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-D-Arg (the Pbf)-OH tripeptide fragment
Liquid phase synthesizing method is used, the preparation of the Wella card peptide-amino main chain peptide resin uses solid-phase synthesis, the present invention
The production of Wella card peptide is carried out by solid-liquid combination mode, production efficiency is can be improved, is suitble to scale industrial production.
It obtains after there is Wella card peptide-amino main chain peptide resin of formula (a) structure, the present invention is by the Wella card peptide-ammonia
Base main chain peptide resin and Mmt protection solution mixing is gone, carries out de- Mmt protection reaction, obtain having the de- Mmt of formula (b) structure to tie up
Draw card peptide-amino main chain peptide resin.In the present invention, it is described go Mmt protection solution be preferably TFA DCM solution, it is described to go
It is preferably 1~3% that Mmt, which protects the volume content of TFA in solution, and more preferably 1.5~2.5%.In the present invention, described de-
The temperature of Mmt protection reaction is preferably 15~30 DEG C, and more preferably 20~25 DEG C；The time of the de- Mmt protection reaction is preferred
For 5~10min, more preferably 6~8min.
After completing de- Mmt protection reaction, obtained product is preferably successively washed and is dried by the present invention, is obtained described
De- Mmt Wella card peptide-amino main chain peptide resin；The washing is preferably DCM and/or DMF with detergent.
It obtains after there is de- Mmt Wella card peptide-amino main chain peptide resin of formula (b) structure, the present invention ties up the de- Mmt
It is mixed after drawing the activation of card peptide-amino main chain peptide resin Cys side chain thiol with Boc-L-Cys-OH, carries out coupling reaction, obtain
Full guard Wella card peptide-amino resins.In the present invention, the Cys side chain of the de- Mmt Wella card peptide-amino main chain peptide resin
The activation method of sulfydryl is preferred are as follows:
It will be mixed in the de- Mmt Wella card peptide-amino main chain peptide resin with activator, carry out priming reaction, obtain Cys
Wella card peptide-amino main chain peptide resin of side chain thiol activation.In the present invention, the equation of the priming reaction such as formula (d)
It is shown；
In the present invention, the activator is preferably 2,2'-, bis- sulphur, two pyridine；The de- Mmt Wella card peptide-amino main chain
The mass ratio of peptide resin and activator is preferably 3.1:1；The activation time is preferably 2h.Present invention preferably uses the sides of stirring
Formula is mixed.
After the completion of priming reaction, the present invention is preferably successively washed and is dried to gained activation products, obtains the Cys
Wella card peptide-amino main chain peptide resin of side chain thiol activation；It is special that the present invention does not have the washing and dry mode
It is required that using washing well known to those skilled in the art and drying mode.
In the present invention, the Wella card peptide-amino main chain peptide resin and Boc-L-Cys-OH of the Cys side chain thiol activation
Molar ratio be preferably 1:5；The temperature of the coupling reaction is preferably 20~30 DEG C, and more preferably 24~28 DEG C；The coupling
The time of reaction is preferably 3h.After the completion of coupling reaction, the present invention preferably to gained full guard Wella card peptide-amino resins successively
It is washed and is dried, the present invention does not have special requirement to the washing and dry mode, uses those skilled in the art
Well known washing and drying mode.The present invention passes through the Cys side chain mercapto of de- Mmt Wella card peptide-amino main chain peptide resin
Coupling reaction is carried out with Boc-L-Cys-OH again after base activation, can successfully construct the disulfide bond of Wella card peptide.
After obtaining full guard Wella card peptide-amino resins, the present invention is by the full guard Wella card peptide-amino resins and splits
It solves reagent mixing and carries out cracking reaction, obtain Wella card peptide.In the present invention, the lytic reagent is preferably TFA, benzene first sulphur
Ether, TIS, EDT and H2The mixed solvent of O, the in the mixed solvent TFA, thioanisole, TIS, EDT and H2The volume ratio of O is preferred
For 80~90:10~5:5~2:5~2:1, more preferably 84~86:9~6:4~3:1；The lytic reagent and all risk insurance
Protecting Wella card peptide-amino resins volume ratio is preferably 5~10:1, more preferably 6~8:1.In the present invention, the cracking is anti-
The temperature answered is preferably 25 DEG C, and the time of the cracking reaction is preferably 3h.The present invention can be by full guard by cracking reaction
Wella card peptide-amino resins is cracked into amino resins and full guard Wella card peptide, and the side chain for cutting off full guard Wella card peptide is protected
Protect base.
After the completion of cracking reaction, the present invention preferably post-processes gained cracking reaction liquid；The post-processing is preferred are as follows:
It filters the lysate to obtain filtrate, the filtrate is added in the ether of pre-cooling and is successively precipitated and is centrifuged, then uses second
Ether is dried in vacuo after being centrifuged product washing, obtains Wella card peptide solid.In the present invention, the ether of the filtrate and pre-cooling
Volume ratio is 1:5~10；The temperature of the ether of the pre-cooling is preferably 0~15 DEG C.The present invention does not dissolve in second using Wella card peptide
The property of ether, when filtrate is added in the ether of pre-cooling, Wella card peptide can be precipitated to form precipitating, with other liquid impurities point
From.The present invention does not have special requirement to the centrifugation, washing and dry mode, uses side well known to those skilled in the art
Formula.
After obtaining Wella card peptide solid, the present invention preferably purifies gained Wella card peptide solid.In the present invention, institute
The mode for stating purifying is preferably chromatographic purification.In the present invention, the chromatographic purification system for use in carrying is preferably
NOVASEPRP-HPLC system, in the present invention, the chromatography wavelength of the chromatographic purification is preferably 220nm, and chromatographic column is preferred
For reverse phase C18 column, mobile phase is preferably TFA and acetonitrile mixed solution, and the volume content of TFA is preferably in the mixed solution
1%.
After purification, the present invention is preferably successively concentrated and dries to gained purified, obtains Wella card peptide sterling；At this
In invention, the mode of the drying is preferably freeze-dried.
It is described in detail below with reference to preparation method of the embodiment to Wella card peptide provided by the invention, but cannot
They are interpreted as limiting the scope of the present invention.
Embodiment 1
The preparation of Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-D-Arg (Pbf)-OH:
1) synthesis of Fmoc-D-Arg (Pbf)-OSu:
0.1molFmoc-D-Arg (Pbf)-OH and 0.11molHOSu is dissolved in 0.2LTHF, ice-water bath is placed in, is obtained mixed
Close liquid I；0.11molDCC is dissolved in 0.1LTHF, and is added dropwise in mixed liquor I, after the 1h that is added dropwise that the reaction was continued, by temperature
Rise to 25 DEG C, after insulation reaction 3h, by reaction solution filtering, be evaporated, then plus DCM dissolution filter, be evaporated, obtain solid I；Add acetic acid second
Ester recrystallizes after dissolving solid I, obtains Fmoc-D-Arg (Pbf)-OSu.
2) synthesis of Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-OH:
By 0.12molH-D-Arg (Pbf)-OH and 0.12molNa2CO3It is dissolved in 0.2L volume fraction 50%THF/H2O is molten
Liquid obtains mixed liquor I I；Fmoc-D-Arg (Pbf)-OSu is dissolved in THF, and is added dropwise in mixed liquor I I, after 25 DEG C of reaction 8h,
Reaction solution is rotated, 15% citric acid tune pH to 3 of volume fraction is added, is extracted with ethyl acetate 3 times, merges having for 3 extractions
After machine phase, is washed 3 times with saturation NaCl solution, add anhydrous Na2SO4It is dried overnight, solvent evaporated, obtains solid II；Use ethyl acetate
It after solid II is dissolved, recrystallizes, obtains Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-OH.
3) synthesis of Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-OSu:
0.1molFmoc-D-Arg (Pbf)-D-Arg (Pbf)-OH and 0.11molHOSu is dissolved in 2LTHF, as ice
Water-bath obtains mixed liquor I II；0.11molDCC is dissolved in 0.1LTHF, and is added dropwise in mixed liquor I II, it is anti-that continuation is added dropwise
After answering 1h, temperature is risen to 25 DEG C, after insulation reaction 3h；By reaction solution filtering, be evaporated, then plus DCM dissolution filter, be evaporated, obtain
Fmoc-D-Arg(Pbf)-D-Arg(Pbf)-OSu。
4) synthesis of Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-D-Arg (Pbf)-OH:
0.12molH-D-Arg (Pbf)-OH and 0.12molNa2CO3 is dissolved in 0.2L volume fraction 50%THF/H2O is molten
Liquid obtains mixed liquor I V；Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-OSu is dissolved in THF, and is added dropwise in mixed liquor I V, 25
DEG C reaction 8h after, reaction solution is rotated, be added 15% citric acid tune pH to 3 of volume fraction, ethyl acetate extract 3 times, merge 3 times
After the organic phase of extraction, is washed 3 times with saturation NaCl solution, add anhydrous Na2SO4It is dried overnight, solvent evaporated obtains Fmoc-D-
Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-OH。
Embodiment 2
The preparation of Wella card peptide-amino main chain peptide resin:
1) Fmoc amino resins is swollen: taking 20g substitution degree is the RinkAmideMBHA resin of 0.5mmol/g, is added
200mlDCM or DMF is by resin swelling 0.5h, after draining solvent, then with DMF washs resin twice, drains solvent, obtain amino tree
Rouge solid I.
2) Fmoc amino resins takes off Fmoc protection: into amino resins solid I made from step 1), 200ml volume is added
Than the deprotection solution of piperidines and DMF composition for 1:4, Fmoc protection is taken off twice under the conditions of 25 DEG C: de- for the first time
Fmoc protection and second of de- Fmoc protection, taking off Fmoc guard time for the first time is 5min, and second of de- Fmoc guard time is
After 10min, then with DMF amino resins pH to 7 is washed, drains solvent, obtain the amino resins of deprotection；
3) amino acid and peptide fragment activation: respectively by Fmoc-D-Arg (Pbf)-OH, Fmoc-D-Ala-OH, Fmoc-D-
Arg (Pbf)-D-Arg (Pbf)-D-Arg (Pbf)-OH, each 30mmol of Fmoc-D-Ala-OH, N-Ac-D-Cys (Mmt)-OH with
Condensation system: 30mmolHOBT and 30mmolDIC is dissolved with suitable DMF, 5min is reacted under the conditions of 25 DEG C, after obtaining activation
Amino acid and peptide fragment.
4) it is condensed: the amino acid after step 3) activation obtained and the peptide fragment after activation being added to and taken off made from step 2)
Except the condensation reaction 2h of amino acid in the amino resins of Fmoc protecting group, is carried out under the conditions of 25 DEG C, ninhydrin chromogenic reaction is supervised
Control condensation reaction process, gradually condensation activation after protected amino acid and peptide fragment: according to A Bapa peptide backbone peptide sequence from C-terminal to
Full guard amino acid and the peptide fragment sequence that N-terminal is successively condensed are Fmoc-D-Arg (Pbf)-OH, Fmoc-D-Ala-OH, Fmoc-
D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-OH,Fmoc-D-Ala-OH,N-Ac-D-Cys(Mmt)-OH；It is final to tie up
Draw card peptide-amino main chain peptide resin N-Ac-D-Cys (Mmt)-D-Ala-D-Arg (Pbf)-D-Arg (Pbf)-D-Arg (Pbf)-
D-Ala-D-Arg (Pbf)-RinkAmideMBHA resin, resin 37.06g, rate of body weight gain 95%.
Above-mentioned ninhydrin coloration method: taking a small amount of resin as in test tube, and DMF is washed twice, and is separately added into test tube
After one 5% ninhydrin of drop volume score-ethanol solution, 85% phenol of drop volume score-ethanol solution and a drop pyridine, in
After heating 2min under the conditions of 120 DEG C, then after being washed twice with DMF, color of resin is observed.
Embodiment 3
The removing of Mmt protecting group:
Wella card peptide made from embodiment 2-amino main chain peptide resin is added in reaction column, DMF swelling is added
30min takes out DMF, and DMF is added and washes twice, adds DCM and washes twice；400ml2%TFA/DCM solution reaction is added
5min, takes out solution, then adds 400ml5%TFA/DCM solution reaction 2min, resin is washed 3 times with 400mlDCM, then is used
DMF is washed 3 times.Resin shrinkage is drained, and 34.34g resin segment: N-Ac-D-Cys-D-Ala-D-Arg (Pbf)-D-Arg is obtained
(Pbf)-D-Arg (Pbf)-D-Ala-D-Arg (Pbf)-RinkAmideMBHA resin.
Embodiment 4
Full guard Wella card peptide-amino resins synthesis:
11.00g2 is weighed, bis- sulphur of 2'-, two pyridine is dissolved in 400mlDMF, is slowly added to the 34.34g resin of embodiment 3
Segment: N-Ac-D-Cys-D-Ala-D-Arg (Pbf)-D-Arg (Pbf)-D-Arg (Pbf)-D-Ala-D-Arg (Pbf)-
It in RinkAmideMBHA resin, is stirred to react 2 hours, drains reaction solution, resin is washed 6 times with DMF, and resin shrinkage is drained, and is obtained
The peptide resin segment activated to Cys side chain thiol: N-Ac-D-Cys (SPyridine)-D-Ala-D-Arg (Pbf)-D-Arg
(Pbf)-D-Arg (Pbf)-D-Ala-D-Arg (Pbf)-RinkAmideMBHA resin.
It weighs 11.05gBoc-L-Cys-OH and 2.0mlDIPEA to be added in 400mlDMF, is slowly added to N-Ac-D-Cys
(SPyridine)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-
RinkAmideMBHA resin reacts at room temperature 3 hours.Coupling terminates, and drains reaction solution, is washed 6 times with DMF, and resin shrinkage is taken out
It is dry, obtain full guard Wella card peptide-amino resins 35.52g.
Embodiment 5
Product cracking:
The lytic reagent of its 10 times of volumes is added into full guard Wella card peptide-amino resins made from embodiment 4,25
It after reacting 3h under the conditions of DEG C, filters, filtrate is added in the ether of its 10 times of volumes pre-cooling and precipitated, be centrifuged, then use ether
By washing of precipitate four times, vacuum drying obtains Wella card peptide.Wherein lytic reagent is TFA, thioanisole, TIS, EDT and H2O
Mixed solvent, wherein volume ratio are as follows: TFA: thioanisole: TIS:EDT:H2O=90:5:2:2:1.
White Wella card peptide solid 10.32g, thick peptide purity 91.8% are obtained, thick peptide yield is 98.5%.
Embodiment 6
The purifying of thick peptide:
The thick peptide 2.00g of Wella card peptide that Example 5 is prepared, using NOVASEPRP-HPLC system, wavelength
220nm, chromatographic column are reverse phase C18 column, and conventional 0.1%TFA/ acetonitrile is mobile phase purifying, and desalination collects purpose peak fraction, rotation
Turn to be concentrated by evaporation, is lyophilized and obtains white Wella card peptide solid 1.61g, yield 80.5%, through detecting HPLC purity 98.50%,
Total recovery 79.3%.Gained Wella card peptide product mass spectrum is as shown in Figure 1, [M]+1:1048.545, the theory of Wella card peptide are accurate
Molecular weight are as follows: 1047.529, sample mass spectral results are consistent with theoretical molecular weight.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of preparation method of Wella card peptide, which comprises the following steps:
(1) under condensation system effect, using fmoc-protected amino resins as carrier, by full guard amino acid and Fmoc-D-Arg
(Pbf)-D-Arg (Pbf)-D-Arg (Pbf)-OH tripeptide fragment is condensed, and obtains the Wella card with structure shown in formula (a)
Peptide-amino main chain peptide resin；Sequence of the condensation according to Wella card peptide backbone peptide from C-terminal to N-terminal carries out；
The condensation of the full guard amino acid and Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-D-Arg (Pbf)-OH tripeptide fragment
Sequentially are as follows: Fmoc-D-Arg (Pbf)-OH, Fmoc-D-Ala-OH, Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-D-Arg
(Pbf)-OH,Fmoc-D-Ala-OH,N-Ac-D-Cys(Mmt)-OH；
(2) by the Wella card peptide-amino main chain peptide resin and Mmt protection solution is gone to mix, carries out de- Mmt protection reaction, obtains
To de- Mmt Wella card peptide-amino main chain peptide resin with structure shown in formula (b)；
(3) it is mixed after activating the Cys side chain thiol of the de- Mmt Wella card peptide-amino main chain peptide resin with Boc-L-Cys-OH
It closes, carries out coupling reaction, obtain the full guard Wella card peptide-amino resins with structure shown in formula (c)；
(4) the full guard Wella card peptide-amino resins and lytic reagent are mixed and carries out cracking reaction, obtain Wella card peptide.
2. preparation method according to claim 1, which is characterized in that Fmoc-D-Arg (the Pbf)-D-Arg (Pbf)-
D-Arg (Pbf)-OH tripeptide fragment is prepared by following methods:
(I) Fmoc-D-Arg (Pbf)-OH and HOSu is subjected under the action of DCC coupling reaction, obtains Fmoc-D-Arg
(Pbf)-OSu；
(II) Fmoc-D-Arg (Pbf)-OSu and H-D-Arg (Pbf)-OH are coupled under the action of base catalyst
Reaction, obtains Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-OH；
(III) Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-OH and HOSu is subjected to coupling reaction under the action of DCC,
Obtain Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-OSu；
(IV) by the Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-OSu and H-D-Arg (Pbf)-OH base catalyst effect
Lower carry out coupling reaction obtains Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-D-Arg (Pbf)-OH.
3. preparation method according to claim 2, which is characterized in that the base catalysis in the step (II) and step (IV)
Agent is Na2CO3。
4. preparation method according to claim 1, which is characterized in that the step (1) specifically:
I) by after the swelling of Fmoc amino resins, de- Fmoc protection is carried out in the case where going Fmoc to protect solution effects, is deprotected
Amino resins；
Ii) by the full guard amino acid, Fmoc-D-Arg (Pbf)-D-Arg (Pbf)-D-Arg (Pbf)-OH tripeptide fragment with
The mixing of condensation system, carries out priming reaction, the amino acid and tripeptide fragment activated；
Iii the amino acid of the activation and tripeptide fragment are mixed with the amino resins of the deprotection), carry out condensation reaction,
Obtain Wella card peptide-amino main chain peptide resin；
The step i) and step ii) not no time sequencing limitation.
5. preparation method according to claim 1 or 4, which is characterized in that the condensation system includes condensing agent and reaction
Solvent, the condensing agent are DIC/HOBt, PyBOP/HOBt/DIEA or HATU/HOBt/DIEA；The reaction dissolvent be DMF,
One or more of DCM, NMP and DMSO；
The molar ratio of the full guard amino acid, condensing agent and Fmoc amino resins is 2~5:2~5:1.
6. preparation method according to claim 1 or 4, which is characterized in that the amino resins be RinkAmide resin,
One or more of RinkAmideMBHA resin and RinkAmideAM resin；The degree of substitution of the amino resins be 0.2~
1.0mmol/g。
7. the preparation method according to claim 4, which is characterized in that in the step i) swelling with sweller be DCM or
DMF；The mixed liquor that Fmoc protection solution is removed as piperidines and DMF.
8. preparation method according to claim 1, which is characterized in that in the step (2) go Mmt protection solution be
The DCM solution of TFA, the volume content for removing TFA in Mmt protection solution is 1~3%.
9. preparation method according to claim 1, which is characterized in that take off Mmt Wella card peptide-amino in the step (3)
The activation method of the Cys side chain thiol of main chain peptide resin are as follows:
The de- Mmt Wella card peptide-amino main chain peptide resin is mixed with activator, priming reaction is carried out, obtains Cys side chain mercapto
Wella card peptide-amino main chain peptide resin of base activation；The activator is bis- sulphur of 2,2'-, two pyridine.
10. preparation method according to claim 1, which is characterized in that the lytic reagent in the step (4) is TFA, benzene
Methyl sulfide, TIS, EDT and H2The mixed solvent of O, the in the mixed solvent TFA, thioanisole, TIS, EDT and H2The volume ratio of O
For 80~90:10~5:5~2:5~2:1.
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