CN104974237B - A kind of method of segment method synthesis in solid state ziconotide - Google Patents
A kind of method of segment method synthesis in solid state ziconotide Download PDFInfo
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Abstract
The invention belongs to Peptides Synthesis, are related to a kind of method of segment method synthesis in solid state ziconotide.The technical problems such as that the technical problem to be solved by the present invention is to synthesis cycles existing for existing preparation method is long, thick peptide purity is low, 3 pairs of disulfide bond formation process trivial operations, total recoverys are low.The technical scheme adopted by the invention is as follows: 4 segment peptide [19-25] A, [12-18] B, [6-11] C, [1-5] D of Solid phase synthesis ziconotide are used first；According still further to sequentially successively full guard peptide fragment B, C, D are connected on peptide resin A, cracking obtains ziconotide linear peptides；Finally by mild condition, operation is simplified, easy post-processing, and environmentally friendly liquid phase one-step oxidation obtains ziconotide.This technical solution can greatly shorten synthesis cycle, reduce synthesis, purifying difficulty and production cost, improve the total recovery of linear peptides purity and product；Conducive to industrial mass production.
Description
Technical field
The present invention relates to Peptides Synthesis, in particular to a kind of method of segment method synthesis in solid state ziconotide.
Background technique
Ziconotide (Ziconotide), trade name Prialt are developed, on January 11st, 2005 by Irish Elan company
For the first time in the granted listing in the U.S., acquisition European Union license in 2 months 2005,2007, Combinated easing pain meeting panel recommended to examine together
Promise peptide is as the intrathecal antalgesic of a line.It is a kind of N-type calcium channel blocker, is divided out of pyramid type snail mollusk body
The synthesized form of the omega-conotoxin M VII A separated out can be used for the nonopioid analgesic object of intrathecal administration.Ziconotide is logical
It crosses special and is effectively combined N-VSCC (N-type voltage-sensitive calcium channels) to block induced pain
The release of mediator, and then realize its pharmacotoxicological effect is suitable for being suitble to intrathecal injection and other treatment method (such as systemic town
Pain medicine, adjuvant treatment or intrathecal morphine) it is not resistant to or the treatment of the adult patient of invalid significant chronic pain, it compares
In opioid analgesic drug, there is good effect, have no drug resistance and without the advantages such as additive.
Ziconotide by 3 pairs of disulfide bond be connected in the polypeptide that forms of 25 amino acid, molecular weight 2639.12,
Peptide sequence is longer and structure is complicated, and C1-C16, C8-C20 and C15-C25 tri- makes polypeptide be folded into ω knot disulfide bond, except this it
It is outer further to stablize its space structure there are also 3 strands of β-pleated sheets.Its amino acid sequence and disulfide bond bridge mode are as shown below:
Synthetic method report about ziconotide is less, and external related patents are concentrated mainly on pharmacology and treatment side
Face；The country about its preparation patent such as: CN200710301598.8, CN200910188686.0, CN201110139661.9,
CN201310202216.1 is all made of solid phase synthesis process, using single fmoc-protected amino acid as raw material, successively connects one by one
Amino acid is synthesized；In patent CN201310202089.5 the synthesis of linear peptides used Fmoc-Ser (tBu)-Gly-OH,
Totally 3 kinds of dipeptide fragments avoid [+Gly] as raw material by Fmoc-Thr (tBu)-Gly-OH, Fmoc-Lys (Boc)-Gly-OH
The generation of ziconotide and [- Gly]-ziconotide impurity.But it is long all to there is synthesis cycle in the above solid phase synthesis process, thick peptide
Purity and the lower problem of yield.
About in ziconotide synthesis step aoxidize form disulfide bond method, patent CN200710301598.8,
CN201110139661.9 and CN201310202216.1 discloses the Cys synthetic thread using 3 kinds of sulfhydryl protected bases of difference
Property peptide the preparation strategy of three pairs of disulfide bond is formed using step-by-step oxidation using the self stability of protecting group；Although formed two
Sulfide linkage directionality is good, but this tactful intermediate steps is more, cumbersome.Patent CN200910188686.0 is same using step oxidation
When formed three pairs of disulfide bond preparation strategy, although simplifying technique, remove Acm used in metal salt compound to people
Body harmfulness is big, is also unfavorable for environmental protection.Iodine, H are disclosed in patent CN201310202089.52O2Deng as oxidant, oxidation shape
At disulfide bond, but this kind of oxidant easily causes the oxidation of Met in ziconotide peptide chain.
Above technical scheme has the disadvantage that during synthesizing peptide chain using the side that amino acid is coupled one by one
Method, synthesis cycle is long, and linear thick peptide purity is low；There are trivial operations during disulfide bond formation, it is difficult to realize that scale is amplified
Production, the low technical problem of total recovery.
Summary of the invention
The present invention provides a kind of method of segment method synthesis in solid state ziconotide, can greatly shorten synthesis cycle and
The purity and yield of thick peptide are improved, reduces purifying difficulty, and oxidizing condition is mild, step is simplified, and convenient for operation, is suitable for work
Industry production.
For achieving the above object, the present invention the following technical schemes are provided:
A kind of method of segment method synthesis in solid state ziconotide, which comprises the steps of:
(1) it uses amino resins for solid phase carrier, then sequentially 7 Fmoc protected amino acids of Leie time coupling, obtains side chain guarantor
Ziconotide segment A [19-25] peptide resin of shield；
Ziconotide segment A [19-25] peptide resin are as follows:
H-Ser (tBu)-Cys (Trt)-Arg (pbf)-Ser (tBu)-Gly-Lys (Boc)-Cys (Trt)-amino resins；
(2) it uses CTC resin for solid phase carrier, successively carries out condensation reaction from C-terminal to N-terminal according to following sequence and obtain side
Ziconotide segment B [12-18] peptide resin, segment C [6-11] peptide resin and segment D [5-1] peptide resin of chain full guard；
Ziconotide segment B [12-18] peptide resin are as follows:
Fmoc-Met-Tyr (tBu)-Asp (OtBu)-Cys (Trt)-Cys (Trt)-Thr (tBu)-Gly-CTC resin；
Ziconotide segment C [6-11] peptide resin are as follows:
Fmoc-Ala-Lys (Boc)-Cys (Trt)-Ser (tBu)-Arg (pbf)-Leu-CTC resin；
Ziconotide segment D [1-5] peptide resin are as follows:
Fmoc- Cys (Trt)-Lys (Boc)-Gly-Lys (Boc)-Gly-CTC resin；
(3) peptide resin of segment B, C, D are cracked, obtain full guard peptide fragment B, C, D, structure are as follows:
Ziconotide segment B [12-18] are as follows:
Fmoc-Met-Tyr(tBu)-Asp(OtBu)-Cys(Trt)-Cys(Trt)-Thr(tBu)-Gly-OH；
Ziconotide segment C [6-11] are as follows:
Fmoc-Ala-Lys(Boc)-Cys(Trt)-Ser(tBu)-Arg(pbf)-Leu-OH；
Ziconotide segment D [1-5] are as follows:
Fmoc- Cys(Trt)-Lys(Boc)-Gly-Lys(Boc)-Gly-OH；
(4) ziconotide segment A [19-25] peptide resin and full guard peptide fragment B connect peptide in the presence of condensing agent
Reaction, generates the peptide resin of 14 amino acid, is sequentially connected full guard peptide fragment C and D according still further to same operation, finally removes
Fmoc completes the preparation of the linear peptide resin of ziconotide.
The structure of the linear peptide resin of ziconotide is as follows:
(5) peptide resin cracks to obtain the linear peptides crude product of ziconotide through lytic reagent；
It (6) will be after the dissolution of the buffered aqueous solution of the linear peptides ammonium acetate of ziconotide and EDTA；Auxiliary oxidizing agent is added,
It adjusts solution ph to stand at low temperature at 7.5 ~ 8.5,2 ~ 8 DEG C to react, HPLC monitors reaction end, obtains examining together containing disulfide bond
Promise peptide crude product；
(7) ziconotide crude product solution pH value is adjusted to be prepared chromatogram purification after 3-6, freeze-drying obtains ziconotide essence
Peptide.
Preferably, step (1) amino resins is one in Rink Amide resin and Rink Amide-AM resin
Kind, the substitution degree of preferred resin is 0.2 ~ 0.5mmol/g.
Preferably, the substitution degree of CTC resin described in step (2) is 0.6 ~ 1.2mmol/g.
Preferably, full guard peptide resin lytic reagent group is combined into TFE/DCM, TFA/DCM, AcOH/TFE/ in step (3)
One of DCM, HFIP/DCM.
Preferably, the lytic reagent of linear peptides described in step (5) is that the TFA of addition volume ratio 1-20% scavenger is molten
Liquid, the scavenger are one in methyl phenyl ethers anisole, thioanisole, dithioglycol, mercaptoethanol, phenol, water and tri isopropyl silane
Kind is several.
Preferably, the specific steps of the liquid phase oxidation of linear peptides described in step (5) are as follows:
The buffered aqueous solution of ammonium acetate and EDTA is prepared, linear peptides dissolution is added, adds auxiliary oxidizing agent, adjusts pH value
Stand at low temperature is reacted to 7.5 ~ 8.5,2 ~ 8 DEG C, and HPLC monitors reaction process.
More preferably, the concentration that linear peptides are controlled in the liquid phase oxidation system of linear peptides is 0.5 ~ 2mg/ml, ammonium acetate
Concentration be 0.5 ~ 0.7mol/L, the concentration of EDTA is 0.5-1.5mmol/L.
Auxiliary oxidizing agent group is combined into GSH (reduced glutathione)/GSSG (oxidized form of glutathione) and cysteine/Guang
One of propylhomoserin；GSH (is gone back in preferred oxidant combination GSH (reduced glutathione)/GSSG (oxidized form of glutathione)
Prototype glutathione) addition concentration be 0.1-10mmol/L, the addition concentration of GSSG (oxidized form of glutathione) is 0.05-
1mmol/L；Preferably, the addition concentration of cysteine is 0.1-10mmol/L, Guang in oxidant combination cysteine/cystine
The addition concentration of propylhomoserin is 0.05-1mmol/L.
Compared with the existing technology, the beneficial effects of the present invention are:
The present invention uses segment method synthesis in solid state ziconotide linear peptides, and 4 segments can be simultaneously synthesizing, substantially reduces
Synthesis cycle.Synthesis difficulty is reduced, making the thick peptide purity of linear peptides is more than 80%；The total recovery of product is improved to 50% or more.It reduces
Purifying difficulty, making the purity of final products is more than 99.5%, further reduced purifying cost.The oxidation side of linear peptides simultaneously
Method has mild condition, and operation is simplified, easy post-processing, environmentally friendly feature, is conducive to industrial mass production.
Specific embodiment
With specific embodiment, the present invention is described in detail below, but protection scope of the present invention is not limited to that；
The feed ratio of feed change, reaction dissolvent or condensing agent etc. according to the present invention, are within the scope of the invention.
Abbreviation meaning used in specification and claims is as follows:
Fmoc 9-fluorenylmethyloxycarbonyl
Acm acetamide methyl
TBu tert-butyl
Pbf 2,2,4,6,7- pentamethyl benzofuran -5- sulfonyl
Trt trityl
Boc tertbutyloxycarbonyl
CTC resin 2- chlorine trityl chloride resin
DCM methylene chloride
DMF N,N-dimethylformamide
DIPEA N, N- diisopropylethylamine
DIC N, N- diisopropylcarbodiimide
HBTU benzotriazole-N, N, N', N'- tetramethylurea hexafluorophosphate
HATU 2- (7- azo benzotriazole)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester
TBTU O- benzotriazole-N, N, N', N'- tetramethylurea tetrafluoro boric acid
HOBT I-hydroxybenzotriazole
HOAT 1- hydroxyl -7- azo benzotriazole
TFA trifluoroacetic acid
TFE trifluoroethanol
EDTA ethylenediamine tetra-acetic acid
GSH reduced glutathione
GSSG oxidized form of glutathione
HFIP hexafluoroisopropanol
The synthesis of embodiment 1:Fmoc- Cys (Trt)-Rink Amide Resins
It accurately weighs Rink Amide resin 20.0g (sub=0.33mmol/g) to be put into synthesis column, with 160mL DMF
It washes twice, 160mL DCM is added and is swollen 30min, after leaching out DCM, is washed twice with 160mL DMF；Be added 20% piperidines/
DMF solution 160ml is deprotected 2 times, reacts 10min and 15min respectively；Then 2 are washed respectively with 100ml DMF, DCM, DMF
It is secondary；DMF is leached out, the mixing DMF solution that Fmoc-Cys (Trt)-OH/DIC/HOBT is added [weighs 7.73g (13.2mmol)
Fmoc- Cys (Trt)-OH and 1.96g (14.52mmol) HOBT is placed in conical flask, and it is molten that the stirring of 100mL DMF solution is added
It solves, 2.25ml (14.52mmol) DIC is added under low temperature (0 DEG C), activate 3min]；2h is reacted, reaction solution is taken out, uses 160mL
DMF is washed twice, and capping reagent 120mL (24ml acetic anhydride, 20.4ml pyridine, 75.6mL DCM) is added and reacts 2h, leaches out
Reaction solution is washed 3 times with DMF, DCM, methanol respectively, and Fmoc-Cys (Trt)-Rink Amide resins is obtained after vacuum drying
22.06g；A small amount of resin is taken, survey substitution degree is 0.29mmol/g.
The synthesis of embodiment 2:Fmoc- Cys (Trt)-Rink Amide-AM Resins
It accurately weighs Rink Amide-AM resin 20.0g (sub=0.32mmol/g) to be put into synthesis column, uses 160mL
DMF is washed twice, and 160mL DCM is added and is swollen 30min, after leaching out DCM, is washed twice with 160mL DMF；It is added 20%
Piperidines/DMF solution 160ml is deprotected 2 times, reacts 10min and 15min respectively；Then it is washed respectively with 100ml DMF, DCM, DMF
It washs 2 times；DMF is leached out, the mixing DMF solution that Fmoc-Cys (Trt)-OH/DIC/HOBT is added [weighs 3.75g (6.4mmol)
Fmoc- Cys (Trt)-OH and 0.95g (7.04mmol) HOBT is placed in conical flask, and it is molten that the stirring of 100mL DMF solution is added
It solves, 1.09ml (7.04mmol) DIC is added under low temperature (0 DEG C), activate 3min]；2h is reacted, reaction solution is taken out, uses 160mL
DMF is washed twice, and capping reagent 120mL (24ml acetic anhydride, 20.4ml pyridine, 75.6mL DCM) is added and reacts 2h, leaches out
Reaction solution is washed 3 times with DMF, DCM, methanol respectively, and Fmoc-Cys (Trt)-Rink Amide-AM is obtained after vacuum drying
resins 24.30g；A small amount of resin is taken, survey substitution degree is 0.18mmol/g.
The synthesis of embodiment 3:Fmoc-Gly-CTC Resins
It weighs CTC resin 70.0g (sub=1.00mmol/g) to be placed in synthesis column, is washed twice, added with 420mL DMF
Enter 420mL DCM swelling 30min；After leaching out DCM, the DCM solution 250ml dissolved with 41.62g Fmoc-Gly-OH is added, it is low
DIPEA 25.45ml is added under warm (0 DEG C), reacts 60min, takes out reaction solution, DCM/CH is added3OH/DIPEA (volume ratio
17:2:1) mixed solution 420ml blocks 30min；Then it is washed respectively 3 times with DMF, DCM, methanol, is dried in vacuo to obtain Fmoc-
Gly-CTC Resins 86.50g；Survey substitution degree is 0.80mmol/g.
The synthesis of embodiment 4:Fmoc-Leu-CTC Resins
It weighs CTC resin 30.0g (sub=1.10mmol/g) to be placed in synthesis column, is washed twice, added with 180mL DMF
Enter 180mL DCM swelling 30min；After leaching out DCM, the DCM solution 100ml dissolved with 23.32g Fmoc-Leu-OH is added, it is low
DIPEA12.00ml is added under warm (0 DEG C), reacts 60min, takes out reaction solution, DCM/CH is added3OH/DIPEA (volume ratio 17:
2:1) mixed solution 180ml blocks 30min；Then it is washed respectively 3 times with DMF, DCM, methanol, is dried in vacuo to obtain Fmoc-Leu-
CTC Resins 39.90g.Survey substitution degree is 0.82mmol/g.
Embodiment 5: the preparation of ziconotide [19-25] peptide resin A
Accurately weighing substitution degree in embodiment 1 is 0.29mmol/g Fmoc-Cys (Trt)-Rink Amide resins
20.69g (synthesis scale 6mmol) is placed in synthesis column, and 120ml DCM is added and is swollen 30min, after leaching out DCM, 120ml
DMF is washed 2 times；20% piperidines/DMF solution 160ml is added to be deprotected 2 times, reacts 10min and 15min respectively；Then 120ml is used
DMF, DCM, DMF are washed 2 times respectively；Fmoc-Lys (Boc)-OH 5.62g (12mmol), HOBT 1.78g is added
The DMF solution 100ml of (13.2mmol) and DIC2.04ml (13.2mmol), drum N2It is stirred to react 2h, reaction end is with Kaiser
Subject to reagent testing result, after reaction reaches terminal, reaction solution is taken out, is washed respectively 2 times with 120ml DMF, DCM, DMF；Then
It is deprotected again.Circulate operation repeatedly, sequentially connected protected amino acid are as follows: Fmoc-Gly-OH, Fmoc-Ser (tBu)-
OH,Fmoc-Arg(pbf)-OH,Fmoc-Cys(Trt)-OH,Fmoc-Ser(tBu)-OH.After prepared by peptide resin, 120ml is used
DCM and methanol wash 3 times respectively, are dried in vacuo to obtain peptide resin 28.56g.
Embodiment 6: the preparation of ziconotide [19-25] peptide resin A
Accurately weighing substitution degree in embodiment 2 is 0.18mmol/g Fmoc-Cys (Trt)-Rink Amide-AM
Resins 16.67g (synthesis scale 3mmol) is placed in synthesis column, and 100ml DCM is added and is swollen 30min, after leaching out DCM,
100ml DMF is washed 2 times；20% piperidines/DMF solution 120ml is added to be deprotected 2 times, reacts 10min and 15min respectively；Then
It is washed respectively 2 times with 100ml DMF, DCM, DMF；Fmoc-Lys (Boc)-OH 2.81g (6mmol), HOBT 0.89g is added
The DMF solution 80ml of (6.6mmol) and DIC1.02ml (6.6mmol), drum N2It is stirred to react 2h, reaction end is tried with Kaiser
Subject to agent testing result, after reaction reaches terminal, reaction solution is taken out, is washed respectively 2 times with 100ml DMF, DCM, DMF；Then again
Deprotection.Circulate operation repeatedly, sequentially connected protected amino acid are as follows: Fmoc-Gly-OH, Fmoc-Ser (tBu)-OH,
Fmoc-Arg(pbf)-OH,Fmoc-Cys(Trt)-OH,Fmoc-Ser(tBu)-OH.After prepared by peptide resin, with 100ml DCM
It is washed respectively with methanol 3 times, is dried in vacuo to obtain peptide resin 20.43g.
Embodiment 7: the preparation of ziconotide [12-18] peptide resin B
Accurately weighing substitution degree in embodiment 3 is 0.80mmol/g Fmoc-Gly-CTC Resins 37.50g (synthesis rule
Mould 30mmol) it is placed in synthesis column, 240ml DCM is added and is swollen 30min, after leaching out DCM, 240ml DMF is washed 2 times；Add
Enter 20% piperidines/DMF solution 240ml to be deprotected 2 times, reacts 10min and 15min respectively；Then 240ml DMF, DCM, DMF are used
It washs 2 times respectively；Be added Fmoc- Thr (tBu)-OH 23.85g (60mmol), HOBT 8.92g (66mmol) and
The DMF solution 160ml of DIC10.22ml (66mmol), drum N2It is stirred to react 2h, reaction end is with Kaiser reagent testing result
Subject to, after reaction reaches terminal, reaction solution is taken out, is washed respectively 2 times with 240ml DMF, DCM, DMF；Then it is deprotected again.So
Iterative cycles operation, sequentially connected protected amino acid are as follows: Fmoc-Cys (Trt)-OH, Fmoc-Cys (Trt)-OH, Fmoc-
Asp(OtBu)-OH,Fmoc-Tyr(tBu)-OH,Fmoc-Met-OH.After prepared by peptide resin, with 240ml DCM and methanol point
Xi Di not be 3 times, it is dried in vacuo to obtain peptide resin 77.32g.
Embodiment 8: the preparation of ziconotide [6-11] peptide resin C
Accurately weighing substitution degree in embodiment 4 is 0.82mmol/g Fmoc-Leu-CTC Resins 36.58g (synthesis rule
Mould 30mmol) it is placed in synthesis column, 240ml DCM is added and is swollen 30min, after leaching out DCM, 240ml DMF is washed 2 times；Add
Enter 20% piperidines/DMF solution 240ml to be deprotected 2 times, reacts 10min and 15min respectively；Then 240ml DMF, DCM, DMF are used
It washs 2 times respectively；38.93g (60mmol) Fmoc-Arg (pbf)-OH, 8.92g (66mmol) HOBT and 10.22ml is added
The DMF solution 160ml of (66mmol) DIC, drum N2It is stirred to react 2h, reaction end is subject to Kaiser reagent testing result, instead
After terminal should being reached, reaction solution is taken out, is washed respectively 2 times with 240ml DMF, DCM, DMF；Then it is deprotected again.It follows repeatedly
Ring operation, sequentially connected protected amino acid are as follows: Fmoc-Ser (tBu)-OH, Fmoc-Cys (Trt)-OH, Fmoc-Lys
(Boc)-OH,Fmoc-Ala-OH.After prepared by peptide resin, is washed respectively 3 times with 240ml DCM and methanol, be dried in vacuo to obtain peptide
Resin 71.95g.
Embodiment 9: the preparation of ziconotide [1-5] peptide resin D
Accurately weighing substitution degree in embodiment 3 is 0.80mmol/g Fmoc-Gly-CTC Resins 37.50g (synthesis rule
Mould 30mmol) it is placed in synthesis column, 240ml DCM is added and is swollen 30min, after leaching out DCM, 240ml DMF is washed 2 times；Add
Enter 20% piperidines/DMF solution 240ml to be deprotected 2 times, reacts 10min and 15min respectively；Then 240ml DMF, DCM, DMF are used
It washs 2 times respectively；28.12g (60mmol) Fmoc-Lys (Boc)-OH, 8.92g (66mmol) is added) HOBT and 10.22ml
The DMF solution 160ml of (66mmol) DIC, drum N2It is stirred to react 2h, reaction end is subject to Kaiser reagent testing result, instead
After terminal should being reached, reaction solution is taken out, is washed respectively 2 times with 240ml DMF, DCM, DMF；Then it is deprotected again.It follows repeatedly
Ring operation, sequentially connected protected amino acid are as follows: Fmoc-Gly-OH, Fmoc-Lys (Boc)-OH, Fmoc-Gly-OH, Fmoc-
Lys(Boc)-OH ,Fmoc-Cys(Trt)-OH.After prepared by peptide resin, 3 times are washed respectively with 240ml DCM and methanol, very
Empty dry peptide resin 61.89g.
Embodiment 10: the preparation of ziconotide intermediate fragments B, C, D
2.8L mixed solution is prepared according to the ratio that TFE:DCM is 1:4.Respectively by peptide tree obtained in embodiment 7,8,9
Rouge is put into 3 round-bottomed flasks, is separately added into above-mentioned solution according to the ratio of every 1g resin 10ml solution, and reaction 4- is stirred at room temperature
5h.It is filtered to remove resin respectively, after filtrate decompression concentration, with -20 DEG C of methyl tertiary butyl ether(MTBE) being pre-chilled sedimentations, centrifugation, washings 5
Secondary, it is respectively 45.52g, 42.36g, 31.76g that intermediate fragments B, C, D are obtained after dry, yield is respectively 90.93%,
91.02%、91.46%。
Embodiment 11: the preparation of ziconotide linear peptides peptide resin
[19-25] the peptide resin A of 6mmol in embodiment 5 is put into composite tube, the DCM swelling 30min of 180ml is added；
DCM is leached out, is washed 3 times with the DMF of 180ml；20% piperidines/DMF solution 120ml is added to be deprotected 2 times, react respectively 10min and
15min；Then it is washed respectively 2 times with 120ml DMF, DCM, DMF；Intermediate fragments B 30.04g (18mmol), HOAT is added
The DMF solution 80ml of 2.68g (19.8mmol) and DIC 3.07ml (19.8mmol), drum N2It is stirred to react 3-4h, reaction end
Kaiser reagent testing result of being subject to takes out reaction solution, washs 2 respectively with 180ml DMF, DCM, DMF after reaction reaches terminal
It is secondary；Then it is deprotected again.Circulate operation repeatedly is sequentially connected intermediate fragments C and D, sloughs the last one amino acid
Fmoc protecting group is washed 3 times respectively with 180ml DCM and methanol, is dried in vacuo to obtain peptide resin 48.56g.
Embodiment 12: the preparation of ziconotide linear peptides peptide resin
[19-25] the peptide resin A of 3mmol in embodiment 6 is put into composite tube, the DCM swelling 30min of 120ml is added；
DCM is leached out, is washed 3 times with the DMF of 120ml；20% piperidines/DMF solution 120ml is added to be deprotected 2 times, react respectively 10min and
15min；Then it is washed respectively 2 times with 120ml DMF, DCM, DMF；Addition intermediate fragments B 11.45g (9mmol),
The DMF solution 50ml of HOAT1.34g (9.9mmol) and DIC 1.53ml (9.9mmol), drum N2It is stirred to react 3-4h, reaction is eventually
Point is subject to Kaiser reagent testing result, and reaction is taken out reaction solution, washed respectively with 120ml DMF, DCM, DMF up to after terminal
It washs 2 times；Then it is deprotected again.Circulate operation repeatedly is sequentially connected intermediate fragments C and D, sloughs the last one amino acid
Fmoc protecting group, washed respectively 3 times with 120ml DCM and methanol, be dried in vacuo to obtain peptide resin 30.46g.
Embodiment 13: the preparation of ziconotide linear peptides
It weighs 11 gained ziconotide peptide resin 48.56g of embodiment to be placed in 1000ml round-bottomed flask, be added under ice bath
500ml lytic reagent (trifluoracetic acid/phenol/thioanisole/water/dithioglycol=82.5/5/5/5/2.5, volume ratio), N2It protects
It after shield is stirred to react 4h, filters and removes resin, filtrate is slowly poured into the 5L methyl tertiary butyl ether(MTBE) of -20 DEG C of pre-coolings, refrigerator-freezer
Interior standing 1h, the solid being centrifuged are dried in vacuo to obtain thick peptide 17.78g, purity after washing 6 times with methyl tertiary butyl ether(MTBE)
81.23%, yield 112.03%.
Embodiment 14: the preparation of ziconotide linear peptides
It weighs 12 gained ziconotide peptide resin 30.46g of embodiment to be placed in 1000ml round-bottomed flask, be added under ice bath
330ml lytic reagent (trifluoracetic acid/phenol/thioanisole/water/dithioglycol=82.5/5/5/5/2.5, volume ratio), N2It protects
It after shield is stirred to react 4h, filters and removes resin, filtrate is slowly poured into the 3.5L methyl tertiary butyl ether(MTBE) of -20 DEG C of pre-coolings, ice
1h is stood in cabinet, the solid being centrifuged is dried in vacuo to obtain thick peptide 8.65g, purity after washing 6 times with methyl tertiary butyl ether(MTBE)
81.23%, yield 108.94%.
Embodiment 15: the preparation of ziconotide
The ammonium acetate of 0.5mol/LM and 1mmol/L and the buffer solution of EDTA are prepared, the 5.00g in embodiment 13 is added
Ziconotide linear peptides (concentration 2mg/ml) add GSH (reduced glutathione) and GSSG (oxidized form of glutathione),
Making its concentration is respectively 0.5mmol/L and 0.1mmol/L, adjusts pH value to 7.8 with glacial acetic acid and ammonium hydroxide, is put in 2 ~ 8 DEG C of low temperature
Reaction is stood, HPLC monitors reaction end.Purified after reaction, freeze-drying obtains ziconotide fine peptide 3.06g, purity
99.62%, yield 61.20%.
Embodiment 16: the preparation of ziconotide
The ammonium acetate of 0.6mol/L and 1.5mmol/L and the buffer solution of EDTA are prepared, the 5.00g in embodiment 14 is added
Ziconotide linear peptides (concentration 1mg/ml), add cysteine plus cystine make its concentration be respectively 1.0mmol/L and
0.1mmol/L adjusts pH value to 8.2 with glacial acetic acid and ammonium hydroxide, is put in 2 ~ 8 DEG C of stand at low temperature reactions, HPLC monitors reaction end.
Purified after reaction, freeze-drying obtains ziconotide fine peptide 3.22g, purity 99.70%, yield 64.40%.
Claims (6)
1. a kind of method of segment method synthesis in solid state ziconotide, which comprises the steps of:
(1) using amino resins as solid phase carrier, then sequentially 7 Fmoc protected amino acids of Leie time coupling, obtain the neat of side chain protection
Examine promise peptide fragment A [19-25] peptide resin；
Ziconotide segment A [19-25] peptide resin are as follows:
H-Ser (tBu)-Cys (Trt)-Arg (pbf)-Ser (tBu)-Gly-Lys (Boc)-Cys (Trt)-amino resins；
(2) using CTC resin as solid phase carrier, condensation reaction is successively carried out from C-terminal to N-terminal according to following sequence and obtains side chain all risk insurance
Ziconotide segment B [12-18] peptide resin, segment C [6-11] peptide resin and segment D [5-1] peptide resin of shield；
Ziconotide segment B [12-18] peptide resin are as follows:
Fmoc-Met-Tyr (tBu)-Asp (OtBu)-Cys (Trt)-Cys (Trt)-Thr (tBu)-Gly-CTC resin；
Ziconotide segment C [6-11] peptide resin are as follows:
Fmoc-Ala-Lys (Boc)-Cys (Trt)-Ser (tBu)-Arg (pbf)-Leu-CTC resin；
Ziconotide segment D [1-5] peptide resin are as follows:
Fmoc-Cys (Trt)-Lys (Boc)-Gly-Lys (Boc)-Gly-CTC resin；
(3) peptide resin of segment B, C, D are cracked, obtain full guard peptide fragment B, C, D, structure are as follows:
Ziconotide segment B [12-18] are as follows:
Fmoc-Met-Tyr(tBu)-Asp(OtBu)-Cys(Trt)-Cys(Trt)-Thr(tBu)-Gly-OH；
Ziconotide segment C [6-11] are as follows:
Fmoc-Ala-Lys(Boc)-Cys(Trt)-Ser(tBu)-Arg(pbf)-Leu-OH；
Ziconotide segment D [1-5] are as follows:
Fmoc-Cys(Trt)-Lys(Boc)-Gly-Lys(Boc)-Gly-OH；
(4) peptide reaction occurs in the presence of condensing agent for ziconotide segment A [19-25] peptide resin and full guard peptide fragment B, raw
At the peptide resin of 14 amino acid, it is sequentially connected full guard peptide fragment C and D according still further to same operation, finally removes Fmoc, it is complete
At the preparation of the linear peptide resin of ziconotide；
The structure of the linear peptide resin of ziconotide is as follows:
H-Cys(Trt)-Lys(Boc)-Gly-Lys(Boc)-Gly-Ala-Lys(Boc)-Cys(Trt)-Ser(tBu)-Arg
(pbf)-Leu-Met-Tyr(tBu)-Asp(OtBu)-Cys(Trt)-Cys(Trt)-Thr(tBu)-Gly-Ser(tBu)-Cys
(Trt)-Arg (pbf)-Ser (tBu)-Gly-Lys (Boc)-Cys (Trt)-amino resins；
(5) peptide resin cracks to obtain the linear peptides crude product of ziconotide through lytic reagent；
(6) by after the dissolution of the buffered aqueous solution of the linear peptides ammonium acetate of ziconotide and EDTA, auxiliary oxidizing agent is added, adjusts
Solution ph to stand at low temperature at 7.5~8.5,2~8 DEG C is reacted, and HPLC monitors reaction end, obtains the Qi Kaonuo containing disulfide bond
Peptide crude product；
The concentration of ziconotide linear peptides is 0.5~2mg/ml in the step (6), and the concentration of ammonium acetate is 0.5~0.7mol/
L；The concentration of EDTA is 0.5-1.5mmol/L；
Auxiliary oxidizing agent group is combined into GSH (reduced glutathione)/GSSG (oxidized form of glutathione), half in the step (6)
One of cystine/cystine；
GSH (reduced form gluathione in oxidant combination GSH (reduced glutathione)/GSSG (oxidized form of glutathione)
Peptide) addition concentration be 0.1-10mmol/L, the addition concentration of GSSG (oxidized form of glutathione) is 0.05-1mmol/L；
The addition concentration of cysteine is 0.1-10mmol/L in the oxidant combination cysteine/cystine, cystine
Addition concentration is 0.05-1mmol/L；
(7) ziconotide crude product solution pH value is adjusted to be prepared chromatogram purification after 3-6, freeze-drying obtains ziconotide fine peptide.
2. the method for segment method synthesis in solid state ziconotide according to claim 1, which is characterized in that step (1) described ammonia
Base resin is Rink Amide resin or Rink Amide-AM resin, and substitution degree is 0.2~0.5mmol/g.
3. the method for segment method synthesis in solid state ziconotide according to claim 1, it is characterised in that: described in step (2)
CTC resin substitution degree be 0.6~1.2mmol/g.
4. the method for segment method synthesis in solid state ziconotide according to claim 1, it is characterised in that: all risk insurance in step (3)
Shield peptide resin lytic reagent group is combined into one of TFE/DCM, TFA/DCM, AcOH/TFE/DCM, HFIP/DCM.
5. the method for segment method synthesis in solid state ziconotide according to claim 1, which is characterized in that the contracting in step (4)
Mixture are as follows: in DIC/HOBT, DIC/HOAT, TBTU/HOBT/DIPEA, HBTU/HOBT/DIPEA, HATU/HOAT/DIPEA
It is a kind of；De- Fmoc reagent is piperidines/DMF solution that volume content is 15~25%.
6. the method for segment method synthesis in solid state ziconotide according to claim 1, which is characterized in that described in step (5)
The lytic reagents of linear peptides be TFA solution that volume content is 1-20% scavenger, the scavenger is methyl phenyl ethers anisole, benzene first sulphur
One or more of ether, dithioglycol, mercaptoethanol, phenol, water and tri isopropyl silane.
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| CN106008674A (en) * | 2016-08-24 | 2016-10-12 | 希施生物科技（上海）有限公司 | Method for preparing linaclotide |
| CN106496315A (en) * | 2016-10-24 | 2017-03-15 | 合肥国肽生物科技有限公司 | A kind of efficient ziconotide synthesis preparation method |
| CN108250265A (en) * | 2016-12-29 | 2018-07-06 | 北京中科亚光生物科技有限公司 | A kind of new process for synthesizing the polypeptide containing intramolecular disulfide bond |
| CN107056894B (en) * | 2017-05-26 | 2021-07-20 | 济南康和医药科技有限公司 | Method for solid-phase synthesis of ganirelix acetate by fragment method |
| CN108047324A (en) * | 2018-02-01 | 2018-05-18 | 润辉生物技术（威海）有限公司 | A kind of preparation method of omega-conotoxin GVIA |
| CN109021087A (en) * | 2018-09-17 | 2018-12-18 | 滨海吉尔多肽有限公司 | A kind of method that solid liquid phase combination prepares ziconotide |
| CN109106942B (en) * | 2018-09-18 | 2019-11-22 | 深圳瑞健生物科技有限公司 | It is a kind of to pass through the application of the polypeptide of blood-brain barrier in medicine preparation |
| CN112279905A (en) * | 2020-10-10 | 2021-01-29 | 杭州固拓生物科技有限公司 | Preparation process of conus anti-wrinkle agent |
| CN115806607A (en) * | 2022-12-27 | 2023-03-17 | 哈尔滨吉象隆生物技术有限公司 | Site-directed cyclization method of chlorotoxin polypeptide |
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