CN110467666A - A kind of synthetic method of novel amylin - Google Patents
A kind of synthetic method of novel amylin Download PDFInfo
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- CN110467666A CN110467666A CN201910875340.1A CN201910875340A CN110467666A CN 110467666 A CN110467666 A CN 110467666A CN 201910875340 A CN201910875340 A CN 201910875340A CN 110467666 A CN110467666 A CN 110467666A
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- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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Abstract
The invention discloses a kind of synthetic methods of novel amylin, comprising the following steps: S1 prepares protected amino acid NH2‑An-Tbos；S2 prepares resin peptide Wang resin-NH-A1‑A2‑…‑An‑CONH2；S3 prepares polypeptide fragment NH2‑A1‑A2‑…‑An‑CONH2；S4 connects sulfydryl, oxidant is provided, by polypeptide fragment NH2‑A1‑A2‑…‑An‑CONH2In Cys sulfydryl using oxidation formed disulfide bond connect two-by-two, obtain H-A1‑A2‑…‑An‑CONH2(Disulfide bridge:x-y) crude product, wherein x and y is digit of any two sulfydryl in polypeptide；S5 purifies crude product.Its by-product is few, and the yield of polypeptide is high, applied widely.
Description
Technical field
The present invention relates to the synthetic methods of polypeptide, and in particular to a kind of synthetic method of novel amylin.
Background technique
Amylin (IAPP, islet amyloid polypeptide) is made of 37 amino acid residues,
Middle second and the 7th cysteine form disulfide bond, sequence carboxy-terminal amidation.
Amylin is originally found in the islet amyloid sample deposit of type II diabetes people, rear to prove it again
It is present in the pancreas and blood plasma of normal humans and animals, and has and adjust the various biologicals effects such as glycometabolism, is considered as
A kind of new pancreas hormone of β cell is coexisted in insulin.
Amyloid beta deposition is formed by by IAPP, have destroy beta Cell of islet membrane structure, inducing beta cell apoptosis and
Damage the effect of β cell function, it is considered to be one of important pathogenesis of type II diabetes.Aggregation, aggregation to IAPP
The structure of body and its toxic effect research to β cell, not only help the pathogenesis of clear type II diabetes, and
Current research also indicates that the apoptosis for inhibiting the aggregation of IAPP that can effectively reduce β cell, improves the success rate of pancreatic islets transplantation.Therefore,
Amylin has become a target spot with good prospect in type II diabetes treatment.
Diabetes are the endocrine system diseases as caused by human nutrition dysbolism, are basic biochemical special with Persistent hyperglycemia
Sign, has become the fifth-largest cause of the death of the mankind at present, it is considered to be harm is only second to the non-infectious of cardiovascular and cerebrovascular diseases and malignant tumour
Disease.And type II diabetes essential characteristic are as follows: on the basis of insulin resistance, β cell function is damaged.Related II type glycosuria
The pathogenesis of disease not yet illustrates at present.It is found in the postmortem to type patient, the islet tissue of about 95% patient occurs
The deposition of amyloid protein package, and this kind of amyloid beta deposition is in Cooper in 1987 etc. from diabetic's pancreas islet group
Its main component is identified in the Amyloid deposition knitted --- the amylin containing 37 amino acid residues, it is multinomial
Studies have shown that the factors such as the sequence of amylin itself, locating environment can influence its aggregation, and inhibit pancreas islet
The aggregation of amyloid polypeptide all shows high value to clinical applications such as the treatments and pancreatic islets transplantation of type II diabetes.
The amylin production method used currently on the market is traditional chemiluminescent polypeptide solid-phase synthesis.Its with
CTC resin is carrier, synthesizes amylin with symmetric anhydride method and DCC-HOBT condensation method.Although activity can be obtained
Relatively high amylin product, but because the generation of β-pleated sheet structure, the sterling yield pole being finally prepared
Low-purity is not high, it is difficult to industrial amplification production.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of synthetic method of novel amylin, by-products
Few, the yield of polypeptide is high, applied widely.
In order to solve the above-mentioned technical problems, the present invention provides a kind of synthetic method of novel amylin, packets
Include following steps:
S1 prepares protected amino acid NH2-An-Tbos
NH is provided2-An- COOH is prepared into protected amino acid NH using activator2-An- Tbos, wherein NH2-An-
COOH is the amino acid of any one with or without side chain protecting group, and protects including even number with or without side chain
Protect the NH of group2- Cys-COOH, n are the natural number not less than 2；
S2 prepares resin peptide Wang resin-NH-A1-A2-…-An-CONH2
Wang resin is provided, using condensing agent by protected amino acid NH2-An- Tbos is according to final product sequence by N-terminal amino
Direction is connected in turn on Wang resin to C-terminal carboxyl direction, obtains Wang resin-NH-A1-A2-…-An-CONH2；
S3 prepares polypeptide fragment NH2-A1-A2-…-An-CONH2
Hydrofluoric acid is provided, removes Wang resin-NH-A with it1-A2-…-An-CONH2In all side chain protecting groups simultaneously
The connection with Wang resin is disconnected, NH is obtained2-A1-A2-…-An-CONH2；
S4 connects sulfydryl
Oxidant is provided, by polypeptide fragment NH2-A1-A2-…-An-CONH2In Cys sulfydryl using oxidation formed two
Sulfide linkage connects two-by-two, obtains H-A1-A2-…-An-CONH2(Disulfide bridge:x-y) crude product, wherein x and y is any
Digit of two sulfydryls in polypeptide；
S5 purifies crude product
The already oxidised peptide purification for forming disulfide bond is obtained into H-A1-A2-…-An-CONH2(Disulfide bridge:
X-y) sterling, wherein x and y is digit of any two sulfydryl in polypeptide.
Preferably, the following steps are included:
S1 prepares protected amino acid NH2-An-Tbos
A. by NH2-An- COOH is dissolved in DCM, and rotor is added, and opens magnetic stirring apparatus, 1min is sufficiently stirred, until
NH2-An- COOH is completely dissolved, wherein NH2-An- COOH is the amino acid of any one with or without side chain protecting group,
And the NH including even number with or without side chain protecting group2- Cys-COOH, n are the natural number not less than 2；
B. SiCl4/tBuOH mixture is added afterwards, addition activator is slowly added dropwise, is stirred to react 15min, is spin-dried for obtaining ammonia
Base acid NH2-An-Tbos；
S2 prepares resin peptide Wang resin-NH-A1-A2-…-An-CONH2
C., hydroxy resin Wang Resin is provided, is added into reaction vessel, DCM is added, nitrogen is opened and rushes from bottom to top
Mixed liquid and Wang Resin, and open blade agitators and stir 15 minutes, it is swollen resin sufficiently, a small amount of DCM is during which added
Prevent solution evaporation to dry；Solvent is leached out by sand core after Wang Resin complete swelling, is added into reaction vessel
NH2-A1DCM is added in-Tbos, and the first condensing agent of triphosgene and 3 times of mol amounts is added, adds 10 times of excessive DIEA of mol,
It extracts after reaction 60min, is washed 2 times with DMF, methanol washs 2 times, and DMF is washed 2 times；
D. eluant, eluent is added, stirs 1min, removes TBos protecting group, obtains resin peptide Wang resin-NH-A1-COOH；
E. next protected amino acid NH is added into reaction vessel2-A2Second condensing agent of-Tbos and 3 times of mol amount, then
10 times of excessive DIC of mol are added, extracts after reacting 60min, is washed 2 times with DMF, methanol washs 2 times, and DMF is washed 2 times；
F. step d and e are repeated, NH is made2-A3- Tbos~NH2-AnThe C-terminal of-Tbos is consecutively connected to the N of monoamino-acid
End；The amino acid starting material of last position can use purchase NH2-An-CONH2To complete；Finally obtain Wang resin-NH-A1-
A2-…-An-CONH2；
G. during reacting, an amino acid is completed in every reaction, goes out solid phase load using Acid and Alkali Titration Analysis method accurate calculation
The content of free carboxy, operating method and condition on body are as follows: weigh the enough NaOH/ of the resin peptide of 5mg after the reaction was completed
MeOH aqueous solution makes the-COOH after de- Tbos or the-COOH after condensation be converted into Na salt, then sufficiently washing, removes remaining
After NaOH, acid-base titration is carried out to-the COONa on carrier with the HCl solution of normal concentration, calculates the carboxylic that dissociates on solid phase carrier
The content of base；
S3 prepares polypeptide fragment NH2-A1-A2-…-An-CONH2
H. by Wang resin-NH-A1-A2-…-An-CONH2It is placed in a beaker, hydrofluoric acid is added under ice bath state, often
Stirring cracking 3H under temperature state；
I. resin is filtered out by sand core afterwards, the ice ether of 10 times of volumes is added, settles 2H after stirring, is placed in
It in 3600 revs/min of centrifuge, is cleaned with ether, takes out sediment, be dried under reduced pressure and do not stop to roll, it is dry until obtaining white
Powdery does not aoxidize disulfide bond polypeptide fragment NH2-A1-A2-…-An-CONH2；
S4 connects sulfydryl
J. by NH2-A1-A2-…-An-CONH2It is dissolved in oxidant, 1~4H of magnetic agitation, is during which tried using ELLMAN
Agent is monitored, and until no longer there are free sulfhydryl groups in product, i.e. the sulfydryl of two Cys of expression utilizes disulfide bond successful connection,
Obtain H-A1-A2-…-An-CONH2(Disulfide bridge:x-y) crude product, wherein x and y is any two sulfydryl more
Digit in peptide；
S5 purifies crude product
K. by the already oxidised polypeptide H-A for forming disulfide bond1-A2-…-An-CONH2(Disulfide bridge:x-y) is thick
Product are added in the mixed solution of pure water and acetonitrile, and according to parts by weight, pure water: acetonitrile 8:2, supersonic oscillations stirring is until complete
Fully dissolved, and passed through filtering with microporous membrane；
L. a liquid chromatography purification: using 0.1%TFA solution as mobile phase A, acetonitrile is Mobile phase B, preparative liquid chromatography
System, using 10 μm reverse phase C18 (100 × 650mm) filled column, UV detector sets 220nm, adjusts flow velocity 80ml/min,
With 5% acetonitrile, balance 15 minutes, sample introduction；Using gradient elution: 0-2min, 5%-5%；2-42min, 17%-23%；42-
48min, 50%-50%；Appearance time is before and after 26min, respectively before collection peak, three sections behind summit, peak, summit collection liquid purity
Greater than 98%, after the collection liquid before peak and behind peak is concentrated into right amount, purity is obtained greater than 98% using same method purified pool
Refined solution, merges collection liquid of all contents 98% or more, and spin concentration removes the acetonitrile in collection liquid；
M. two liquid chromatography purifications: using injection pure water as mobile phase A, acetonitrile is Mobile phase B；Prepare preparation liquid phase color
Spectra system, using 10 μm of reverse phase C18 (100 × 650mm) filled columns, UV detector sets 220nm, adjusts flow velocity 60ml/min,
First with 80% acetonitrile, balances 10 minutes, then with 2% acetonitrile, balance 15 minutes；A liquid chromatography purification is taken to obtain
Purpose peptide collection liquid, sample introduction, using gradient elution: 0-15min, 5%-5%；15-65min, 5%-40% are opened when main peak occurs
Begin to collect, until terminating when occurring without peak, merge collection liquid, spin concentration removes the acetonitrile and water in collection liquid, and refined liquid is made；
N. refined liquid is dispensed in multiple clean eggplant-shape bottles, refined liquid is frozen on eggplant-shape bottle wall using liquid nitrogen, merging
The H-A of high-purity is arrived in freeze dryer1-A2-…-An-CONH2(Disulfide bridge:x-y) sterling.
Preferably, activator in step a is n,N-diisopropylethylamine, N, N- diisopropyl carbon two is sub- and pyridine
One or more of.
Preferably, the eluant, eluent in step d is 10%TFA+90%DCM mixed solution, 5%TFA+95%DCM is mixed
Close one or more of solution and 20% piperidines+80%DCM mixed solution.
Preferably, the first condensing agent in step c is 2- (7- azo benzotriazole)-N, N, N', N'- tetramethyl
Urea hexafluorophosphoric acid ester, 1H- benzotriazole -1- oxygen tripyrrole quinoline drone hexafluorophosphoric acid and (7- azepine benzotriazole -1- oxygen) tripyrrole
One or more of phosphorus hexafluorophosphate.
Preferably, the second condensing agent in step c is I-hydroxybenzotriazole, 1- hydroxyl -7- azepine benzotriazole
One or more of with 4- dimethylamino pyridine.
Preferably, the second condensing agent in step e is I-hydroxybenzotriazole, 1- hydroxyl -7- azepine benzotriazole
One or more of with 4- dimethylamino pyridine.
Preferably, the oxidant in step j is one or more of water, DMSO, iodine, trifluoroacetic acid.
Preferably, the oxidant in step j is the mixed solution of DMSO and water, according to parts by weight, its ratio be
1:9.
Preferably, the following steps are included:
S1 prepares protected amino acid NH2-Lys(Boc)-Tbos、NH2-Cys(Trt)-Tbos、NH2-Asn(Trt)-
Tbos、NH2-Thr(Tbu)-Tbos、NH2-Ala-Tbos、NH2-Gln(Trt)-Tbos、NH2-Arg(Pbf)-Tbos、NH2-
Leu-Tbos、NH2-Phe-Tbos、NH2-Val-Tbos、NH2-His(Boc)-Tbos、NH2-Ser(Tbu)-Tbos、NH2-Gly-
Tbos、NH2-Ile-Tbos
A. by the NH of 50g2- Lys (Boc)-Tbos is added into 2.5L beaker, is dissolved in the DCM of 2L, and rotor is added,
Magnetic stirring apparatus is opened, 1min is sufficiently stirred, until NH2- Lys (Boc)-Tbos is completely dissolved；
B. the t-BuOH mixture of the SiCl4 and 3g of 6.9g are added afterwards, is slowly added dropwise and pyridine 10.8ml is added, be stirred to react
15min is spin-dried for obtaining amino acid N H2- Lys (Boc)-Tbos, according to above method successively by NH2-Cys(Trt)-COOH、NH2-
Asn(Trt)-COOH、NH2-Thr(Tbu)-COOH、NH2-Ala-COOH、NH2-Gln(Trt)-COOH、NH2-Arg(Pbf)-
COOH、NH2-Leu-COOH、NH2-Phe-COOH、NH2-Val-COOH、NH2-His(Boc)-COOH、NH2-Ser(Tbu)-
COOH、NH2-Gly-COOH、NH2- Ile-COOH is prepared into protected amino acid NH2-Lys(Boc)-Tbos、NH2-Cys(Trt)-
Tbos、NH2-Asn(Trt)-Tbos、NH2-Thr(Tbu)-Tbos、NH2-Ala-Tbos、NH2-Gln(Trt)-Tbos、NH2-Arg
(Pbf)-Tbos、NH2-Leu-Tbos、NH2-Phe-Tbos、NH2-Val-Tbos、NH2-His(Boc)-Tbos、NH2-Ser
(Tbu)-Tbos、NH2-Gly-Tbos、NH2-Ile-Tbos；
S2 prepares resin peptide
Wangresin-NH-Lys(Boc)-Cys(Trt)-Asn(Trt)-Thr(Tbu)-Ala-Thr(Tbu)-Cys
(Trt)-Ala-Thr(Tbu)-Gln(Trt)-Arg(Pbf)-Leu-Ala-Asn(Trt)-Phe-Leu-Val-His(Boc)-
Ser(Tbu)-Ser(Tbu)-Asn(Trt)-Asn(Trt)-Phe-Gly-Ala-Ile-Leu-Ser(Tbu)-Ser(Tbu)-Thr
(Tbu)-Asn(Trt)-Val-Gly-Ser(Tbu)-Asn(Trt)-Thr(Tbu)-Tyr(Tbu)-CONH2
C., the 100g hydroxy resin Wang Resin that substitution degree is 0.3mmol/g is provided, is added into reaction vessel, is added
500mlDCM opens nitrogen and rushes mixed liquid and Wang Resin from bottom to top, and opens blade agitators and stir 15 minutes, makes to set
Rouge is sufficiently swollen, and a small amount of DCM, which is during which added, prevents solution evaporation to dry；It is filtered after Wang Resin complete swelling by sand core
Fall solvent, NH is added into reaction vessel2- Lys (Boc)-Tbos, is added the DCM of 500ml, be added 26.6g triphosgene and
The HATU of 26.6g adds 10 times of excessive DIEA of mol, extracts after reacting 60min, washs 2 times with the DMF of 500ml, 50ml
Methanol wash 2 times, the DMF of 50ml is washed 2 times；
D. the total 500ml of 10%TFA+90%DCM mixed solution is added, stirs 1min, removes TBos protecting group, obtains resin
Peptide Wang resin-NH-Lys (Boc)-COOH；
E. protected amino acid NH is added into reaction vessel2The HOBT of-Cys (Trt)-Tbos and 12.1g, adds 10 times
The excessive DIC of mol is extracted after reacting 60min, is washed 2 times with the DMF of 500ml, and the methanol of 500ml washs 2 times, 500ml's
DMF is washed 2 times；
F. repeat step d and e, make NH2-Asn (Trt)-Tbos, NH2-Thr (Tbu)-Tbos, NH2-Ala-Tbos,
NH2-Gln(Trt)-Tbos、NH2-Arg(Pbf)-Tbos、NH2-Leu-Tbos、NH2-Phe-Tbos、NH2-Val-Tbos、
NH2-His (Boc)-Tbos, NH2-Ser (Tbu)-Tbos, NH2-Gly-Tbos, NH2-Ile-Tbos C-terminal be consecutively connected to
The N-terminal of upper monoamino-acid；The amino acid starting material of last position can use purchase NH2-Tyr(Tbu)-CONH2To complete；Final
To Wangresin-NH-Lys (Boc)-Cys (Trt)-Asn (Trt)-Thr (Tbu)-Ala-Thr (Tbu)-Cys (Trt)-Ala-
Thr(Tbu)-Gln(Trt)-Arg(Pbf)-Leu-Ala-Asn(Trt)-Phe-Leu-Val-His(Boc)-Ser(Tbu)-Ser
(Tbu)-Asn(Trt)-Asn(Trt)-Phe-Gly-Ala-Ile-Leu-Ser(Tbu)-Ser(Tbu)-Thr(Tbu)-Asn
(Trt)-Val-Gly-Ser(Tbu)-Asn(Trt)-Thr(Tbu)-Tyr(Tbu)-CONH2；
G. during reacting, an amino acid is completed in every reaction, goes out solid phase load using Acid and Alkali Titration Analysis method accurate calculation
The content of free carboxy, operating method and condition on body are as follows: weigh the enough NaOH/ of the resin peptide of 5mg after the reaction was completed
MeOH aqueous solution makes the-COOH after de- Tbos or the-COOH after condensation be converted into Na salt, then sufficiently washing, removes remaining
After NaOH, acid-base titration is carried out to-the COONa on carrier with the HCl solution of normal concentration, calculates the carboxylic that dissociates on solid phase carrier
The content of base；
S3 prepares polypeptide fragment NH2-A1-A2-…-An-CONH2
H. by the Wangresin-NH-Lys of 320g (Boc)-Cys (Trt)-Asn (Trt)-Thr (Tbu)-Ala-Thr
(Tbu)-Cys(Trt)-Ala-Thr(Tbu)-Gln(Trt)-Arg(Pbf)-Leu-Ala-Asn(Trt)-Phe-Leu-Val-
His(Boc)-Ser(Tbu)-Ser(Tbu)-Asn(Trt)-Asn(Trt)-Phe-Gly-Ala-Ile-Leu-Ser(Tbu)-Ser
(Tbu)-Thr(Tbu)-Asn(Trt)-Val-Gly-Ser(Tbu)-Asn(Trt)-Thr(Tbu)-Tyr(Tbu)-CONH2It is placed in
In the beaker of 2.5L, the hydrofluoric acid of 3500ml is added under ice bath state, stirring cracking 3H under normal temperature state；
I. resin is filtered out by sand core afterwards, the ice ether of 10 times of volumes is added, settles 2H after stirring, is placed in
It in 3600 revs/min of centrifuge, is cleaned with ether, takes out sediment, be dried under reduced pressure and do not stop to roll, it is dry until obtaining white
Powdery does not aoxidize disulfide bond polypeptide fragment NH2-Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-
Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-Thr-Asn-
Val-Gly-Ser-Asn-Thr-Tyr–CONH2；
S4 connects sulfydryl
J. by 109g polypeptide fragment
NH2-Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-
Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-Thr-Asn-Val-Gly-Ser-Asn-
Thr-Tyr–CONH2It is dissolved in the mixed solution of 10%DMSO and water, 1~4H of magnetic agitation, during which utilizes ELLMAN reagent
It is monitored, until no longer there are free sulfhydryl groups in product, that is, indicates that the sulfydryl of two Cys utilizes disulfide bond successful connection, obtain
It arrives
H-Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-
Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-Thr-Asn-Val-Gly-Ser-Asn-
Thr-Tyr–CONH2(Disulfide bridge:2-7) crude product；
S5 purifies crude product
K. by the already oxidised polypeptide for forming disulfide bond
H-Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-
Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-Thr-Asn-Val-Gly-Ser-Asn-
Thr-Tyr–CONH2(Disulfide bridge:2-7) crude product is added in the mixed solution of 1500ml pure water and acetonitrile, by weight
Measure number meter, pure water: acetonitrile 8:2, supersonic oscillations stirring is until be completely dissolved, and it is passed through 0.45um miillpore filter mistake
Filter；
L. a liquid chromatography purification: using 0.1%TFA solution as mobile phase A, acetonitrile is Mobile phase B, preparative liquid chromatography
System, using 10 μm reverse phase C18 (100 × 650mm) filled column, UV detector sets 220nm, adjusts flow velocity 80ml/min,
With 5% acetonitrile, balance 15 minutes, sample introduction；Using gradient elution: 0-2min, 5%-5%；2-42min, 17%-23%；42-
48min, 50%-50%；Appearance time is before and after 26min, respectively before collection peak, three sections behind summit, peak, summit collection liquid purity
Greater than 98%, after the collection liquid before peak and behind peak is concentrated into right amount, purity is obtained greater than 98% using same method purified pool
Refined solution, merges collection liquid of all contents 98% or more, and spin concentration removes the acetonitrile in collection liquid；
M. two liquid chromatography purifications: using injection pure water as mobile phase A, acetonitrile is Mobile phase B；Prepare preparation liquid phase color
Spectra system, using 10 μm of reverse phase C18 (100 × 650mm) filled columns, UV detector sets 220nm, adjusts flow velocity 60ml/min,
First with 80% acetonitrile, balances 10 minutes, then with 2% acetonitrile, balance 15 minutes；A liquid chromatography purification is taken to obtain
Purpose peptide collection liquid, sample introduction, using gradient elution: 0-15min, 5%-5%；15-65min, 5%-40% are opened when main peak occurs
Begin to collect, until terminating when occurring without peak, merge collection liquid, spin concentration removes the acetonitrile and water in collection liquid, and refined liquid is made；
N. refined liquid is dispensed in multiple clean eggplant-shape bottles, refined liquid is frozen on eggplant-shape bottle wall using liquid nitrogen, merging
To get arriving high-purity in freeze dryer
H-Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-
Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-Thr-Asn-Val-Gly-Ser-Asn-
Thr-Tyr–CONH2(Disulfidebridge:2-7) sterling.
Compared with prior art, the beneficial effects of the present invention are:
1, the present invention carries out the synthesis of amylin using N-terminal-C-terminal synthesis sequence, smoothly avoids pancreas islet
The difficult problem of amyloid polypeptide synthesis, the purifying of β-pleated sheet structure is simple, yield is high.
2, the present invention can be suitable for a variety of polypeptide sequences for being difficult to synthesize, applied widely.
3, the present invention avoids the major site of β-pleated sheet structure formation by rational routes, improves bonding degree, reduces secondary
The chance that product is formed, substantially increases the yield of polypeptide.
Detailed description of the invention
It, below will be in embodiment technical description for the clearer technical solution illustrated in technology of the embodiment of the present invention
Required attached drawing is briefly described, it should be apparent that, the accompanying drawings in the following description is only some realities of the invention
Example is applied, for those of ordinary skill in the art, without creative efforts, additionally it is possible to according to these attached drawings
Obtain other attached drawings.
Fig. 1 is the structural formula figure of amylin；
Fig. 2 is the preparation method figure of protected amino acid used in amylin in embodiment 2；
Fig. 3 is the HPLC analysis chart of amylin sterling in embodiment 2；
Fig. 4 is the mass spectral analysis figure of amylin sterling in embodiment 2.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Whole description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment during this is practical, those of ordinary skill in the art are obtained all without creative labor
Other embodiments shall fall within the protection scope of the present invention.
Embodiment 1
Present embodiment discloses a kind of synthetic methods of novel amylin, comprising the following steps:
S1 prepares protected amino acid NH2-An-Tbos
NH is provided2-An- COOH is prepared into protected amino acid NH using activator2-An- Tbos, wherein NH2-An-
COOH is the amino acid of any one with or without side chain protecting group, and protects including even number with or without side chain
Protect the NH of group2- Cys-COOH, n are the natural number not less than 2.
S2 prepares resin peptide Wang resin-NH-A1-A2-…-An-CONH2
Wang resin is provided, using condensing agent by protected amino acid NH2-An- Tbos is according to final product sequence by N-terminal amino
Direction is connected in turn on Wang resin to C-terminal carboxyl direction, obtains Wang resin-NH-A1-A2-…-An-CONH2。
S3 prepares polypeptide fragment NH2-A1-A2-…-An-CONH2
Hydrofluoric acid is provided, removes Wang resin-NH-A with it1-A2-…-An-CONH2In all side chain protecting groups simultaneously
The connection with Wang resin is disconnected, NH is obtained2-A1-A2-…-An-CONH2。
S4 connects sulfydryl
Oxidant is provided, by polypeptide fragment NH2-A1-A2-…-An-CONH2In Cys sulfydryl using oxidation formed two
Sulfide linkage connects two-by-two, obtains H-A1-A2-…-An-CONH2(Disulfide bridge:x-y) crude product, wherein x and y is any
Digit of two sulfydryls in polypeptide.
S5 purifies crude product
The already oxidised peptide purification for forming disulfide bond is obtained into H-A1-A2-…-An-CONH2(Disulfide bridge:
X-y) sterling, wherein x and y is digit of any two sulfydryl in polypeptide.
Specifically, the following steps are included:
S1 prepares protected amino acid NH2-An-Tbos
A. by NH2-An- COOH is dissolved in DCM, and rotor is added, and opens magnetic stirring apparatus, 1min is sufficiently stirred, until
NH2-An- COOH is completely dissolved, wherein NH2-An- COOH is the amino acid of any one with or without side chain protecting group,
And the NH including even number with or without side chain protecting group2- Cys-COOH, n are the natural number not less than 2；
B. SiCl4/tBuOH mixture is added afterwards, addition activator is slowly added dropwise, is stirred to react 15min, is spin-dried for obtaining ammonia
Base acid NH2-An-Tbos。
S2 prepares resin peptide Wang resin-NH-A1-A2-…-An-CONH2
C., hydroxy resin Wang Resin is provided, is added into reaction vessel, DCM is added, nitrogen is opened and rushes from bottom to top
Mixed liquid and Wang Resin, and open blade agitators and stir 15 minutes, it is swollen resin sufficiently, a small amount of DCM is during which added
Prevent solution evaporation to dry；Solvent is leached out by sand core after Wang Resin complete swelling, is added into reaction vessel
NH2-A1DCM is added in-Tbos, and the first condensing agent of triphosgene and 3 times of mol amounts is added, adds 10 times of excessive DIEA of mol,
It extracts after reaction 60min, is washed 2 times with DMF, methanol washs 2 times, and DMF is washed 2 times；
D. eluant, eluent is added, stirs 1min, removes TBos protecting group, obtains resin peptide Wang resin-NH-A1-COOH；
E. next protected amino acid NH is added into reaction vessel2-A2Second condensing agent of-Tbos and 3 times of mol amount, then
10 times of excessive DIC of mol are added, extracts after reacting 60min, is washed 2 times with DMF, methanol washs 2 times, and DMF is washed 2 times；
F. step d and e are repeated, NH is made2-A3- Tbos~NH2-AnThe C-terminal of-Tbos is consecutively connected to the N of monoamino-acid
End；The amino acid starting material of last position can use purchase NH2-An-CONH2To complete；Finally obtain Wang resin-NH-A1-
A2-…-An-CONH2；
G. during reacting, an amino acid is completed in every reaction, goes out solid phase load using Acid and Alkali Titration Analysis method accurate calculation
The content of free carboxy, operating method and condition on body are as follows: weigh the enough NaOH/ of the resin peptide of 5mg after the reaction was completed
MeOH aqueous solution makes the-COOH after de- Tbos or the-COOH after condensation be converted into Na salt, then sufficiently washing, removes remaining
After NaOH, acid-base titration is carried out to-the COONa on carrier with the HCl solution of normal concentration, calculates the carboxylic that dissociates on solid phase carrier
The content of base.
S3 prepares polypeptide fragment NH2-A1-A2-…-An-CONH2
H. by Wang resin-NH-A1-A2-…-An-CONH2It is placed in a beaker, hydrofluoric acid is added under ice bath state, often
Stirring cracking 3H under temperature state；
I. resin is filtered out by sand core afterwards, the ice ether of 10 times of volumes is added, settles 2H after stirring, is placed in
It in 3600 revs/min of centrifuge, is cleaned with ether, takes out sediment, be dried under reduced pressure and do not stop to roll, it is dry until obtaining white
Powdery does not aoxidize disulfide bond polypeptide fragment NH2-A1-A2-…-An-CONH2。
S4 connects sulfydryl
J. by NH2-A1-A2-…-An-CONH2It is dissolved in oxidant, 1~4H of magnetic agitation, is during which tried using ELLMAN
Agent is monitored, and until no longer there are free sulfhydryl groups in product, i.e. the sulfydryl of two Cys of expression utilizes disulfide bond successful connection,
Obtain H-A1-A2-…-An-CONH2(Disulfide bridge:x-y) crude product, wherein x and y is any two sulfydryl more
Digit in peptide.
S5 purifies crude product
K. by the already oxidised polypeptide H-A for forming disulfide bond1-A2-…-An-CONH2(Disulfide bridge:x-y) is thick
Product are added in the mixed solution of pure water and acetonitrile, and according to parts by weight, pure water: acetonitrile 8:2, supersonic oscillations stirring is until complete
Fully dissolved, and passed through filtering with microporous membrane；
L. a liquid chromatography purification: using 0.1%TFA solution as mobile phase A, acetonitrile is Mobile phase B, preparative liquid chromatography
System, using 10 μm reverse phase C18 (100 × 650mm) filled column, UV detector sets 220nm, adjusts flow velocity 80ml/min,
With 5% acetonitrile, balance 15 minutes, sample introduction；Using gradient elution: 0-2min, 5%-5%；2-42min, 17%-23%；42-
48min, 50%-50%；Appearance time is before and after 26min, respectively before collection peak, three sections behind summit, peak, summit collection liquid purity
Greater than 98%, after the collection liquid before peak and behind peak is concentrated into right amount, purity is obtained greater than 98% using same method purified pool
Refined solution, merges collection liquid of all contents 98% or more, and spin concentration removes the acetonitrile in collection liquid；
M. two liquid chromatography purifications: using injection pure water as mobile phase A, acetonitrile is Mobile phase B；Prepare preparation liquid phase color
Spectra system, using 10 μm of reverse phase C18 (100 × 650mm) filled columns, UV detector sets 220nm, adjusts flow velocity 60ml/min,
First with 80% acetonitrile, balances 10 minutes, then with 2% acetonitrile, balance 15 minutes；A liquid chromatography purification is taken to obtain
Purpose peptide collection liquid, sample introduction, using gradient elution: 0-15min, 5%-5%；15-65min, 5%-40% are opened when main peak occurs
Begin to collect, until terminating when occurring without peak, merge collection liquid, spin concentration removes the acetonitrile and water in collection liquid, and refined liquid is made；
N. refined liquid is dispensed in multiple clean eggplant-shape bottles, refined liquid is frozen on eggplant-shape bottle wall using liquid nitrogen, merging
The H-A of high-purity is arrived in freeze dryer1-A2-…-An-CONH2(Disulfide bridge:x-y) sterling.
As further improvement of this embodiment, the activator in above-mentioned steps a is n,N-diisopropylethylamine, N, N- bis-
One or more of two Asia of isopropyl carbon and pyridine.
As further improvement of this embodiment, the eluant, eluent in above-mentioned steps d is that 10%TFA+90%DCM mixing is molten
One or more of liquid, 5%TFA+95%DCM mixed solution and 20% piperidines+80%DCM mixed solution.
As further improvement of this embodiment, the first condensing agent in above-mentioned steps c is 2- (three nitrogen of 7- azo benzo
Azoles)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester, 1H- benzotriazole -1- oxygen tripyrrole quinoline drone hexafluorophosphoric acid and (7- azepine
One or more of benzotriazole -1- oxygen) tripyrrole phosphorus hexafluorophosphate.
As further improvement of this embodiment, the second condensing agent in above-mentioned steps e is I-hydroxybenzotriazole, 1- hydroxyl
One or more of base -7- azepine benzotriazole and 4- dimethylamino pyridine.
As further improvement of this embodiment, the oxidant in above-mentioned steps j is water, in DMSO, iodine, trifluoroacetic acid
It is one or more of.
As further improvement of this embodiment, the oxidant in above-mentioned steps j is the mixed solution of DMSO and water, by weight
Number meter is measured, its ratio be 1:9.
Embodiment 2
As shown in Figure 1, present embodiment discloses a kind of synthetic methods of novel amylin comprising following step
It is rapid:
S1 prepares protected amino acid NH2-Lys(Boc)-Tbos、NH2-Cys(Trt)-Tbos、NH2-Asn(Trt)-
Tbos、NH2-Thr(Tbu)-Tbos、NH2-Ala-Tbos、NH2-Gln(Trt)-Tbos、NH2-Arg(Pbf)-Tbos、NH2-
Leu-Tbos、NH2-Phe-Tbos、NH2-Val-Tbos、NH2-His(Boc)-Tbos、NH2-Ser(Tbu)-Tbos、NH2-Gly-
Tbos、NH2-Ile-Tbos
A. by the NH of 50g2- Lys (Boc)-Tbos is added into 2.5L beaker, is dissolved in the DCM of 2L, and rotor is added,
Magnetic stirring apparatus is opened, 1min is sufficiently stirred, until NH2- Lys (Boc)-Tbos is completely dissolved；
B. the t-BuOH mixture of the SiCl4 and 3g of 6.9g are added afterwards, is slowly added dropwise and pyridine 10.8ml is added, be stirred to react
15min is spin-dried for obtaining amino acid N H2- Lys (Boc)-Tbos, according to above method successively by NH2-Cys(Trt)-COOH、NH2-
Asn(Trt)-COOH、NH2-Thr(Tbu)-COOH、NH2-Ala-COOH、NH2-Gln(Trt)-COOH、NH2-Arg(Pbf)-
COOH、NH2-Leu-COOH、NH2-Phe-COOH、NH2-Val-COOH、NH2-His(Boc)-COOH、NH2-Ser(Tbu)-
COOH、NH2-Gly-COOH、NH2- Ile-COOH is prepared into protected amino acid NH2-Lys(Boc)-Tbos、NH2-Cys(Trt)-
Tbos、NH2-Asn(Trt)-Tbos、NH2-Thr(Tbu)-Tbos、NH2-Ala-Tbos、NH2-Gln(Trt)-Tbos、NH2-Arg
(Pbf)-Tbos、NH2-Leu-Tbos、NH2-Phe-Tbos、NH2-Val-Tbos、NH2-His(Boc)-Tbos、NH2-Ser
(Tbu)-Tbos、NH2-Gly-Tbos、NH2-Ile-Tbos。
S2 prepares resin peptide
Wangresin-NH-Lys(Boc)-Cys(Trt)-Asn(Trt)-Thr(Tbu)-Ala-Thr(Tbu)-Cys
(Trt)-Ala-Thr(Tbu)-Gln(Trt)-Arg(Pbf)-Leu-Ala-Asn(Trt)-Phe-Leu-Val-His(Boc)-
Ser(Tbu)-Ser(Tbu)-Asn(Trt)-Asn(Trt)-Phe-Gly-Ala-Ile-Leu-Ser(Tbu)-Ser(Tbu)-Thr
(Tbu)-Asn(Trt)-Val-Gly-Ser(Tbu)-Asn(Trt)-Thr(Tbu)-Tyr(Tbu)-CONH2
C., the 100g hydroxy resin Wang Resin that substitution degree is 0.3mmol/g is provided, is added into reaction vessel, is added
500mlDCM opens nitrogen and rushes mixed liquid and Wang Resin from bottom to top, and opens blade agitators and stir 15 minutes, makes to set
Rouge is sufficiently swollen, and a small amount of DCM, which is during which added, prevents solution evaporation to dry；It is filtered after Wang Resin complete swelling by sand core
Fall solvent, NH is added into reaction vessel2- Lys (Boc)-Tbos, is added the DCM of 500ml, be added 26.6g triphosgene and
The HATU of 26.6g adds 10 times of excessive DIEA of mol, extracts after reacting 60min, washs 2 times with the DMF of 500ml, 50ml
Methanol wash 2 times, the DMF of 50ml is washed 2 times；
D. the total 500ml of 10%TFA+90%DCM mixed solution is added, stirs 1min, removes TBos protecting group, obtains resin
Peptide Wang resin-NH-Lys (Boc)-COOH；
E. protected amino acid NH is added into reaction vessel2The HOBT of-Cys (Trt)-Tbos and 12.1g, adds 10 times
The excessive DIC of mol is extracted after reacting 60min, is washed 2 times with the DMF of 500ml, and the methanol of 500ml washs 2 times, 500ml's
DMF is washed 2 times；
F. repeat step d and e, make NH2-Asn (Trt)-Tbos, NH2-Thr (Tbu)-Tbos, NH2-Ala-Tbos,
NH2-Gln(Trt)-Tbos、NH2-Arg(Pbf)-Tbos、NH2-Leu-Tbos、NH2-Phe-Tbos、NH2-Val-Tbos、
NH2-His (Boc)-Tbos, NH2-Ser (Tbu)-Tbos, NH2-Gly-Tbos, NH2-Ile-Tbos C-terminal be consecutively connected to
The N-terminal of upper monoamino-acid；The amino acid starting material of last position can use purchase NH2-Tyr(Tbu)-CONH2To complete；Final
To Wangresin-NH-Lys (Boc)-Cys (Trt)-Asn (Trt)-Thr (Tbu)-Ala-Thr (Tbu)-Cys (Trt)-Ala-
Thr(Tbu)-Gln(Trt)-Arg(Pbf)-Leu-Ala-Asn(Trt)-Phe-Leu-Val-His(Boc)-Ser(Tbu)-Ser
(Tbu)-Asn(Trt)-Asn(Trt)-Phe-Gly-Ala-Ile-Leu-Ser(Tbu)-Ser(Tbu)-Thr(Tbu)-Asn
(Trt)-Val-Gly-Ser(Tbu)-Asn(Trt)-Thr(Tbu)-Tyr(Tbu)-CONH2；
G. during reacting, an amino acid is completed in every reaction, goes out solid phase load using Acid and Alkali Titration Analysis method accurate calculation
The content of free carboxy, operating method and condition on body are as follows: weigh the enough NaOH/ of the resin peptide of 5mg after the reaction was completed
MeOH aqueous solution makes the-COOH after de- Tbos or the-COOH after condensation be converted into Na salt, then sufficiently washing, removes remaining
After NaOH, acid-base titration is carried out to-the COONa on carrier with the HCl solution of normal concentration, calculates the carboxylic that dissociates on solid phase carrier
The content of base.
S3 prepares polypeptide fragment NH2-A1-A2-…-An-CONH2
H. by the Wangresin-NH-Lys of 320g (Boc)-Cys (Trt)-Asn (Trt)-Thr (Tbu)-Ala-Thr
(Tbu)-Cys(Trt)-Ala-Thr(Tbu)-Gln(Trt)-Arg(Pbf)-Leu-Ala-Asn(Trt)-Phe-Leu-Val-
His(Boc)-Ser(Tbu)-Ser(Tbu)-Asn(Trt)-Asn(Trt)-Phe-Gly-Ala-Ile-Leu-Ser(Tbu)-Ser
(Tbu)-Thr(Tbu)-Asn(Trt)-Val-Gly-Ser(Tbu)-Asn(Trt)-Thr(Tbu)-Tyr(Tbu)-CONH2It is placed in
In the beaker of 2.5L, the hydrofluoric acid of 3500ml is added under ice bath state, stirring cracking 3H under normal temperature state；
I. resin is filtered out by sand core afterwards, the ice ether of 10 times of volumes is added, settles 2H after stirring, is placed in
It in 3600 revs/min of centrifuge, is cleaned with ether, takes out sediment, be dried under reduced pressure and do not stop to roll, it is dry until obtaining white
Powdery does not aoxidize disulfide bond polypeptide fragment NH2-Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-
Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-Thr-Asn-
Val-Gly-Ser-Asn-Thr-Tyr–CONH2。
S4 connects sulfydryl
J. by 109g polypeptide fragment
NH2-Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-
Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-Thr-Asn-Val-Gly-Ser-Asn-
Thr-Tyr–CONH2It is dissolved in the mixed solution of 10%DMSO and water, 1~4H of magnetic agitation, during which utilizes ELLMAN reagent
It is monitored, until no longer there are free sulfhydryl groups in product, that is, indicates that the sulfydryl of two Cys utilizes disulfide bond successful connection, obtain
It arrives
H-Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-
Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-Thr-Asn-Val-Gly-Ser-Asn-
Thr-Tyr–CONH2(Disulfide bridge:2-7) crude product.
S5 purifies crude product
K. by the already oxidised polypeptide for forming disulfide bond
H-Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-
Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-Thr-Asn-Val-Gly-Ser-Asn-
Thr-Tyr–CONH2(Disulfide bridge:2-7) crude product is added in the mixed solution of 1500ml pure water and acetonitrile, by weight
Measure number meter, pure water: acetonitrile 8:2, supersonic oscillations stirring is until be completely dissolved, and it is passed through 0.45um miillpore filter mistake
Filter；
L. a liquid chromatography purification: using 0.1%TFA solution as mobile phase A, acetonitrile is Mobile phase B, preparative liquid chromatography
System, using 10 μm reverse phase C18 (100 × 650mm) filled column, UV detector sets 220nm, adjusts flow velocity 80ml/min,
With 5% acetonitrile, balance 15 minutes, sample introduction；Using gradient elution: 0-2min, 5%-5%；2-42min, 17%-23%；42-
48min, 50%-50%；Appearance time is before and after 26min, respectively before collection peak, three sections behind summit, peak, summit collection liquid purity
Greater than 98%, after the collection liquid before peak and behind peak is concentrated into right amount, purity is obtained greater than 98% using same method purified pool
Refined solution, merges collection liquid of all contents 98% or more, and spin concentration removes the acetonitrile in collection liquid；
M. two liquid chromatography purifications: using injection pure water as mobile phase A, acetonitrile is Mobile phase B；Prepare preparation liquid phase color
Spectra system, using 10 μm of reverse phase C18 (100 × 650mm) filled columns, UV detector sets 220nm, adjusts flow velocity 60ml/min,
First with 80% acetonitrile, balances 10 minutes, then with 2% acetonitrile, balance 15 minutes；A liquid chromatography purification is taken to obtain
Purpose peptide collection liquid, sample introduction, using gradient elution: 0-15min, 5%-5%；15-65min, 5%-40% are opened when main peak occurs
Begin to collect, until terminating when occurring without peak, merge collection liquid, spin concentration removes the acetonitrile and water in collection liquid, and refined liquid is made；
N. refined liquid is dispensed in multiple clean eggplant-shape bottles, refined liquid is frozen on eggplant-shape bottle wall using liquid nitrogen, merging
To get arriving high-purity in freeze dryer
H-Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-
Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-Thr-Asn-Val-Gly-Ser-Asn-
Thr-Tyr–CONH2(Disulfidebridge:2-7) sterling.
The chemical formula of amylin are as follows:
H-Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-
Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-Thr-Asn-Val-Gly-Ser-Asn-
Thr-Tyr–CONH2(Disulfidebridge:2-7)。
Product analysis
Referring to shown in Fig. 3, after amylin sterling polypeptide takes sample to dissolve using pure water, using in embodiment 2
The condition of step m obtains the HPLC analysis chart of amylin sterling, passes through reference area, it can be seen that the area of main peak
Accounting has reached 98.2%, then proves that the purity of amylin has reached 98.2%.
Referring to shown in Fig. 4, amylin sterling polypeptide takes sample to dissolve using pure water, ESI cation map
Show that cation trivalent target peak is 1302.2, cation divalent target peak is 1952.8, and product mass spectrum is correct.
To sum up, the amylin total yield of products of the present embodiment synthesis reaches 74.72%, remote unconventional and current
Chemical synthesis means on the market.
It should be noted that some common abbreviations of the present invention have following meanings:
Wang resin: Wang resin
Tbos: butyl silicate
DMSO: dimethyl sulfoxide
DCM: methylene chloride
HATU:2- (7- aoxidizes benzotriazole)-N, N, N', N'- tetramethylurea hexafluorophosphate
DIEA:N, N- diisopropylethylamine
DMF:N, dinethylformamide
HOBT:1- hydroxybenzotriazole
The abbreviation of DIC:N, N'- diisopropylcarbodiimide
TFA: trifluoroacetic acid
The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention.
Various modifications to these embodiments will be readily apparent to those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, of the invention
It is not intended to be limited to the embodiments shown herein, and is to fit to consistent with principles disclosed herein and novel point
Widest scope.
Claims (10)
1. a kind of synthetic method of novel amylin, which comprises the following steps:
S1 prepares protected amino acid NH2-An-Tbos
NH is provided2-An- COOH is prepared into protected amino acid NH using activator2-An- Tbos, wherein NH2-An- COOH is
The amino acid of any one with or without side chain protecting group, and including even number with or without side chain protecting group
NH2- Cys-COOH, n are the natural number not less than 2；
S2 prepares resin peptide Wang resin-NH-A1-A2-…-An-CONH2
Wang resin is provided, using condensing agent by protected amino acid NH2-An- Tbos is according to final product sequence by N-terminal amino direction
It is connected in turn on Wang resin to C-terminal carboxyl direction, obtains Wang resin-NH-A1-A2-…-An-CONH2；
S3 prepares polypeptide fragment NH2-A1-A2-…-An-CONH2
Hydrofluoric acid is provided, removes Wang resin-NH-A with it1-A2-…-An-CONH2In all side chain protecting groups and disconnect
With the connection of Wang resin, NH is obtained2-A1-A2-…-An-CONH2；
S4 connects sulfydryl
Oxidant is provided, by polypeptide fragment NH2-A1-A2-…-An-CONH2In Cys sulfydryl using oxidation formed disulfide bond
It connects two-by-two, obtains H-A1-A2-…-An-CONH2(Disulfide bridge:x-y) crude product, wherein x and y is any two
Digit of the sulfydryl in polypeptide；
S5 purifies crude product
The already oxidised peptide purification for forming disulfide bond is obtained into H-A1-A2-…-An-CONH2(Disulfide bridge:x-y) is pure
Product, wherein x and y is digit of any two sulfydryl in polypeptide.
2. the synthetic method of novel amylin as described in claim 1, which comprises the following steps:
S1 prepares protected amino acid NH2-An-Tbos
A. by NH2-An- COOH is dissolved in DCM, and rotor is added, and opens magnetic stirring apparatus, 1min is sufficiently stirred, until NH2-An-
COOH is completely dissolved, wherein NH2-An- COOH is the amino acid of any one with or without side chain protecting group, and including
The NH of even number with or without side chain protecting group2- Cys-COOH, n are the natural number not less than 2；
B. SiCl4/tBuOH mixture is added afterwards, addition activator is slowly added dropwise, is stirred to react 15min, is spin-dried for obtaining amino acid
NH2-An-Tbos；
S2 prepares resin peptide Wang resin-NH-A1-A2-…-An-CONH2
C., hydroxy resin Wang Resin is provided, is added into reaction vessel, DCM is added, nitrogen is opened and rushes mixed liquid from bottom to top
Body and Wang Resin, and open blade agitators and stir 15 minutes, it is swollen resin sufficiently, a small amount of DCM, which is during which added, to be prevented
Solution evaporation is to dry；Solvent is leached out by sand core after Wang Resin complete swelling, NH is added into reaction vessel2-A1-
DCM is added in Tbos, and the first condensing agent of triphosgene and 3 times of mol amounts is added, adds 10 times of excessive DIEA of mol, reacts
It extracts after 60min, is washed 2 times with DMF, methanol washs 2 times, and DMF is washed 2 times；
D. eluant, eluent is added, stirs 1min, removes TBos protecting group, obtains resin peptide Wang resin-NH-A1-COOH；
E. next protected amino acid NH is added into reaction vessel2-A2Second condensing agent of-Tbos and 3 times of mol amount, adds
10 times of excessive DIC of mol are extracted after reacting 60min, are washed 2 times with DMF, and methanol washs 2 times, and DMF is washed 2 times；
F. step d and e are repeated, NH is made2-A3- Tbos~NH2-AnThe C-terminal of-Tbos is consecutively connected to the N-terminal of monoamino-acid；Most
The amino acid starting material of position can use purchase NH afterwards2-An-CONH2To complete；Finally obtain Wang resin-NH-A1-A2-…-
An-CONH2；
G. during reacting, an amino acid is completed in every reaction, is gone out on solid phase carrier using Acid and Alkali Titration Analysis method accurate calculation
The content of free carboxy, operating method and condition are as follows: weigh the resin peptide of 5mg after the reaction was completed with enough NaOH/MeOH water
Solution makes the-COOH after de- Tbos or the-COOH after condensation be converted into Na salt, then sufficiently washing, after removing remaining NaOH,
Acid-base titration is carried out to-the COONa on carrier with the HCl solution of normal concentration, calculates containing for free carboxy on solid phase carrier
Amount；
S3 prepares polypeptide fragment NH2-A1-A2-…-An-CONH2
H. by Wang resin-NH-A1-A2-…-An-CONH2It is placed in a beaker, hydrofluoric acid, normal temperature state is added under ice bath state
Lower stirring cracks 3H；
I. resin is filtered out by sand core afterwards, the ice ether of 10 times of volumes is added, settles 2H after stirring, is placed in 3600
Rev/min centrifuge in, cleaned with ether, take out sediment, be dried under reduced pressure and do not stop to roll, until obtaining white dry powder-shaped
Disulfide bond polypeptide fragment NH is not aoxidized2-A1-A2-…-An-CONH2；
S4 connects sulfydryl
J. by NH2-A1-A2-…-An-CONH2Be dissolved in oxidant, 1~4H of magnetic agitation, during which using ELLMAN reagent into
Row monitoring indicates that the sulfydryl of two Cys utilizes disulfide bond successful connection, obtains until no longer there are free sulfhydryl groups in product
H-A1-A2-…-An-CONH2(Disulfide bridge:x-y) crude product, wherein x and y is any two sulfydryl in polypeptide
Digit；
S5 purifies crude product
K. by the already oxidised polypeptide H-A for forming disulfide bond1-A2-…-An-CONH2(Disulfide bridge:x-y) crude product is added
In the mixed solution of pure water and acetonitrile, according to parts by weight, pure water: acetonitrile 8:2, supersonic oscillations stirring is until completely molten
Solution, and passed through filtering with microporous membrane；
L. a liquid chromatography purification: using 0.1%TFA solution as mobile phase A, acetonitrile is Mobile phase B, preparative liquid chromatography system
System, using 10 μm reverse phase C18 (100 × 650mm) filled column, UV detector sets 220nm, adjusts flow velocity 80ml/min, with
5% acetonitrile balances 15 minutes, sample introduction；Using gradient elution: 0-2min, 5%-5%；2-42min, 17%-23%；42-
48min, 50%-50%；Appearance time is before and after 26min, respectively before collection peak, three sections behind summit, peak, summit collection liquid purity
Greater than 98%, after the collection liquid before peak and behind peak is concentrated into right amount, purity is obtained greater than 98% using same method purified pool
Refined solution, merges collection liquid of all contents 98% or more, and spin concentration removes the acetonitrile in collection liquid；
M. two liquid chromatography purifications: using injection pure water as mobile phase A, acetonitrile is Mobile phase B；Prepare preparative liquid chromatography system
System, using 10 μm of reverse phase C18 (100 × 650mm) filled columns, UV detector sets 220nm, adjusts flow velocity 60ml/min, first with
80% acetonitrile is balanced 10 minutes, then with 2% acetonitrile, is balanced 15 minutes；The purpose for taking a liquid chromatography purification to obtain
Peptide collection liquid, sample introduction, using gradient elution: 0-15min, 5%-5%；15-65min, 5%-40% start to receive when main peak occurs
Collection merges collection liquid until terminating when occurring without peak, and spin concentration removes the acetonitrile and water in collection liquid, and refined liquid is made；
N. refined liquid is dispensed in multiple clean eggplant-shape bottles, refined liquid is frozen on eggplant-shape bottle wall using liquid nitrogen, merging freeze-drying
The H-A of high-purity is arrived in machine1-A2-…-An-CONH2(Disulfide bridge:x-y) sterling.
3. the synthetic method of novel amylin as claimed in claim 2, which is characterized in that the activation in step a
Agent is N, N- diisopropylethylamine, N, one or more of two Asia of N- diisopropyl carbon and pyridine.
4. the synthetic method of novel amylin as claimed in claim 2, which is characterized in that the elution in step d
Agent is 10%TFA+90%DCM mixed solution, 5%TFA+95%DCM mixed solution and 20% piperidines+80%DCM mixed solution
One or more of.
5. the synthetic method of novel amylin as claimed in claim 2, which is characterized in that first in step c
Condensing agent is 2- (7- azo benzotriazole)-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester, 1H- benzotriazole -1- oxygen three
One or more of pyrrolin drone hexafluorophosphoric acid and (7- azepine benzotriazole -1- oxygen) tripyrrole phosphorus hexafluorophosphate.
6. the synthetic method of novel amylin as claimed in claim 2, which is characterized in that second in step c
Condensing agent is one or more of I-hydroxybenzotriazole, 1- hydroxyl -7- azepine benzotriazole and 4- dimethylamino pyridine.
7. the synthetic method of novel amylin as claimed in claim 2, which is characterized in that second in step e
Condensing agent is one or more of I-hydroxybenzotriazole, 1- hydroxyl -7- azepine benzotriazole and 4- dimethylamino pyridine.
8. the synthetic method of novel amylin as claimed in claim 2, which is characterized in that the oxidation in step j
Agent is one or more of water, DMSO, iodine, trifluoroacetic acid.
9. the synthetic method of novel amylin as claimed in claim 2, which is characterized in that the oxidation in step j
Agent is the mixed solution of DMSO and water, and according to parts by weight, its ratio be 1:9.
10. the synthetic method of novel amylin as described in claim 1, which comprises the following steps:
S1 prepares protected amino acid NH2-Lys(Boc)-Tbos、NH2-Cys(Trt)-Tbos、NH2-Asn(Trt)-Tbos、NH2-
Thr(Tbu)-Tbos、NH2-Ala-Tbos、NH2-Gln(Trt)-Tbos、NH2-Arg(Pbf)-Tbos、NH2-Leu-Tbos、
NH2-Phe-Tbos、NH2-Val-Tbos、NH2-His(Boc)-Tbos、NH2-Ser(Tbu)-Tbos、NH2-Gly-Tbos、NH2-
Ile-Tbos
A. by the NH of 50g2- Lys (Boc)-Tbos is added into 2.5L beaker, is dissolved in the DCM of 2L, and rotor is added, and opens magnetic
Power blender, is sufficiently stirred 1min, until NH2- Lys (Boc)-Tbos is completely dissolved；
B. the t-BuOH mixture of the SiCl4 and 3g of 6.9g are added afterwards, is slowly added dropwise and pyridine 10.8ml is added, be stirred to react
15min is spin-dried for obtaining amino acid N H2- Lys (Boc)-Tbos, according to above method successively by NH2-Cys(Trt)-COOH、NH2-
Asn(Trt)-COOH、NH2-Thr(Tbu)-COOH、NH2-Ala-COOH、NH2-Gln(Trt)-COOH、NH2-Arg(Pbf)-
COOH、NH2-Leu-COOH、NH2-Phe-COOH、NH2-Val-COOH、NH2-His(Boc)-COOH、NH2-Ser(Tbu)-
COOH、NH2-Gly-COOH、NH2- Ile-COOH is prepared into protected amino acid NH2-Lys(Boc)-Tbos、NH2-Cys(Trt)-
Tbos、NH2-Asn(Trt)-Tbos、NH2-Thr(Tbu)-Tbos、NH2-Ala-Tbos、NH2-Gln(Trt)-Tbos、NH2-Arg
(Pbf)-Tbos、NH2-Leu-Tbos、NH2-Phe-Tbos、NH2-Val-Tbos、NH2-His(Boc)-Tbos、NH2-Ser
(Tbu)-Tbos、NH2-Gly-Tbos、NH2-Ile-Tbos；
S2 prepares resin peptide
Wangresin-NH-Lys(Boc)-Cys(Trt)-Asn(Trt)-Thr(Tbu)-Ala-Thr(Tbu)-Cys(Trt)-
Ala-Thr(Tbu)-Gln(Trt)-Arg(Pbf)-Leu-Ala-Asn(Trt)-Phe-Leu-Val-His(Boc)-Ser
(Tbu)-Ser(Tbu)-Asn(Trt)-Asn(Trt)-Phe-Gly-Ala-Ile-Leu-Ser(Tbu)-Ser(Tbu)-Thr
(Tbu)-Asn(Trt)-Val-Gly-Ser(Tbu)-Asn(Trt)-Thr(Tbu)-Tyr(Tbu)-CONH2
C., the 100g hydroxy resin Wang Resin that substitution degree is 0.3mmol/g is provided, is added into reaction vessel, is added
500mlDCM opens nitrogen and rushes mixed liquid and Wang Resin from bottom to top, and opens blade agitators and stir 15 minutes, makes to set
Rouge is sufficiently swollen, and a small amount of DCM, which is during which added, prevents solution evaporation to dry；It is filtered after Wang Resin complete swelling by sand core
Fall solvent, NH is added into reaction vessel2- Lys (Boc)-Tbos, is added the DCM of 500ml, be added 26.6g triphosgene and
The HATU of 26.6g adds 10 times of excessive DIEA of mol, extracts after reacting 60min, washs 2 times with the DMF of 500ml, 50ml
Methanol wash 2 times, the DMF of 50ml is washed 2 times；
D. the total 500ml of 10%TFA+90%DCM mixed solution is added, stirs 1min, removes TBos protecting group, obtains resin peptide
Wang resin-NH-Lys(Boc)-COOH；
E. protected amino acid NH is added into reaction vessel2The HOBT of-Cys (Trt)-Tbos and 12.1g adds 10 times of mol mistakes
The DIC of amount is extracted after reacting 60min, is washed 2 times with the DMF of 500ml, and the methanol of 500ml washs 2 times, the DMF washing of 500ml
2 times；
F. step d and e are repeated, NH2-Asn (Trt)-Tbos, NH2-Thr (Tbu)-Tbos, NH2-Ala-Tbos, NH2-Gln are made
(Trt)-Tbos、NH2-Arg(Pbf)-Tbos、NH2-Leu-Tbos、NH2-Phe-Tbos、NH2-Val-Tbos、NH2-His
(Boc)-Tbos, NH2-Ser (Tbu)-Tbos, NH2-Gly-Tbos, NH2-Ile-Tbos C-terminal be consecutively connected to an amino
The N-terminal of acid；The amino acid starting material of last position can use purchase NH2-Tyr(Tbu)-CONH2To complete；It finally obtains
Wangresin-NH-Lys(Boc)-Cys(Trt)-Asn(Trt)-Thr(Tbu)-Ala-Thr(Tbu)-Cys(Trt)-Ala-
Thr(Tbu)-Gln(Trt)-Arg(Pbf)-Leu-Ala-Asn(Trt)-Phe-Leu-Val-His(Boc)-Ser(Tbu)-Ser
(Tbu)-Asn(Trt)-Asn(Trt)-Phe-Gly-Ala-Ile-Leu-Ser(Tbu)-Ser(Tbu)-Thr(Tbu)-Asn
(Trt)-Val-Gly-Ser(Tbu)-Asn(Trt)-Thr(Tbu)-Tyr(Tbu)-CONH2；
G. during reacting, an amino acid is completed in every reaction, is gone out on solid phase carrier using Acid and Alkali Titration Analysis method accurate calculation
The content of free carboxy, operating method and condition are as follows: weigh the resin peptide of 5mg after the reaction was completed with enough NaOH/MeOH water
Solution makes the-COOH after de- Tbos or the-COOH after condensation be converted into Na salt, then sufficiently washing, after removing remaining NaOH,
Acid-base titration is carried out to-the COONa on carrier with the HCl solution of normal concentration, calculates containing for free carboxy on solid phase carrier
Amount；
S3 prepares polypeptide fragment NH2-A1-A2-…-An-CONH2
H. by the Wangresin-NH-Lys of 320g (Boc)-Cys (Trt)-Asn (Trt)-Thr (Tbu)-Ala-Thr (Tbu)-
Cys(Trt)-Ala-Thr(Tbu)-Gln(Trt)-Arg(Pbf)-Leu-Ala-Asn(Trt)-Phe-Leu-Val-His
(Boc)-Ser(Tbu)-Ser(Tbu)-Asn(Trt)-Asn(Trt)-Phe-Gly-Ala-Ile-Leu-Ser(Tbu)-Ser
(Tbu)-Thr(Tbu)-Asn(Trt)-Val-Gly-Ser(Tbu)-Asn(Trt)-Thr(Tbu)-Tyr(Tbu)-CONH2It is placed in
In the beaker of 2.5L, the hydrofluoric acid of 3500ml is added under ice bath state, stirring cracking 3H under normal temperature state；
I. resin is filtered out by sand core afterwards, the ice ether of 10 times of volumes is added, settles 2H after stirring, is placed in 3600
Rev/min centrifuge in, cleaned with ether, take out sediment, be dried under reduced pressure and do not stop to roll, until obtaining white dry powder-shaped
Disulfide bond polypeptide fragment NH is not aoxidized2-Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-
Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-Thr-Asn-Val-
Gly-Ser-Asn-Thr-Tyr–CONH2；
S4 connects sulfydryl
J. by 109g polypeptide fragment
NH2-Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-
His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-Thr-Asn-Val-Gly-Ser-Asn-Thr-
Tyr–CONH2It is dissolved in the mixed solution of 10%DMSO and water, 1~4H of magnetic agitation, is during which carried out using ELLMAN reagent
Monitoring indicates that the sulfydryl of two Cys utilizes disulfide bond successful connection, obtains until no longer there are free sulfhydryl groups in product
H-Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-
His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-Thr-Asn-Val-Gly-Ser-Asn-Thr-
Tyr–CONH2(Disulfide bridge:2-7) crude product；
S5 purifies crude product
K. by the already oxidised polypeptide for forming disulfide bond
H-Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-
His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-Thr-Asn-Val-Gly-Ser-Asn-Thr-
Tyr–CONH2(Disulfide bridge:2-7) crude product is added in the mixed solution of 1500ml pure water and acetonitrile, by weight
Number meter, pure water: acetonitrile 8:2, supersonic oscillations stirring is until be completely dissolved, and it is passed through 0.45um filtering with microporous membrane；
L. a liquid chromatography purification: using 0.1%TFA solution as mobile phase A, acetonitrile is Mobile phase B, preparative liquid chromatography system
System, using 10 μm reverse phase C18 (100 × 650mm) filled column, UV detector sets 220nm, adjusts flow velocity 80ml/min, with
5% acetonitrile balances 15 minutes, sample introduction；Using gradient elution: 0-2min, 5%-5%；2-42min, 17%-23%；42-
48min, 50%-50%；Appearance time is before and after 26min, respectively before collection peak, three sections behind summit, peak, summit collection liquid purity
Greater than 98%, after the collection liquid before peak and behind peak is concentrated into right amount, purity is obtained greater than 98% using same method purified pool
Refined solution, merges collection liquid of all contents 98% or more, and spin concentration removes the acetonitrile in collection liquid；
M. two liquid chromatography purifications: using injection pure water as mobile phase A, acetonitrile is Mobile phase B；Prepare preparative liquid chromatography system
System, using 10 μm of reverse phase C18 (100 × 650mm) filled columns, UV detector sets 220nm, adjusts flow velocity 60ml/min, first with
80% acetonitrile is balanced 10 minutes, then with 2% acetonitrile, is balanced 15 minutes；The purpose for taking a liquid chromatography purification to obtain
Peptide collection liquid, sample introduction, using gradient elution: 0-15min, 5%-5%；15-65min, 5%-40% start to receive when main peak occurs
Collection merges collection liquid until terminating when occurring without peak, and spin concentration removes the acetonitrile and water in collection liquid, and refined liquid is made；
N. refined liquid is dispensed in multiple clean eggplant-shape bottles, refined liquid is frozen on eggplant-shape bottle wall using liquid nitrogen, merging freeze-drying
The H-Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-As n- of high-purity is arrived in machine
Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-Thr-Asn-Val-Gly-
Ser-Asn-Thr-Tyr–CONH2(Disulfidebridge:2-7) sterling.
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