CN103159845A - Method for synthetizing aviptadil - Google Patents

Method for synthetizing aviptadil Download PDF

Info

Publication number
CN103159845A
CN103159845A CN2013101008523A CN201310100852A CN103159845A CN 103159845 A CN103159845 A CN 103159845A CN 2013101008523 A CN2013101008523 A CN 2013101008523A CN 201310100852 A CN201310100852 A CN 201310100852A CN 103159845 A CN103159845 A CN 103159845A
Authority
CN
China
Prior art keywords
resin
fmoc
trt
coupling
polypeptide fragment
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN2013101008523A
Other languages
Chinese (zh)
Other versions
CN103159845B (en
Inventor
潘俊锋
覃亮政
刘建
马亚平
袁建成
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hybio Pharmaceutical Co Ltd
Original Assignee
Hybio Pharmaceutical Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hybio Pharmaceutical Co Ltd filed Critical Hybio Pharmaceutical Co Ltd
Priority to CN201310100852.3A priority Critical patent/CN103159845B/en
Publication of CN103159845A publication Critical patent/CN103159845A/en
Application granted granted Critical
Publication of CN103159845B publication Critical patent/CN103159845B/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Landscapes

  • Peptides Or Proteins (AREA)

Abstract

The invention relates to the field of medicine synthesis and discloses a method for synthetizing aviptadil. The method comprises the steps of according to the amino acid sequence of aviptadil peptide chain from N terminal to C terminal, synthetizing a 1-9 fragment by using Boc-His(3-Bum)-Ser(Psi(Me, Me)pro)-OH, synthetizing 10-18 and 19-28 fragments simultaneously, coupling the three polypeptide fragments to obtain the aviptadil. By adopting the method, the problems that in the conventional synthetic methods, the content of racemic impurities D-Hisl- aviptadil and D-Ser2-aviptadil is higher and the aviptadil purity is lower due to the inappropriate synthetic materials and coupling methods are solved.

Description

A kind of method of synthetic aviptadil
Technical field
The present invention relates to medicine and synthesize the field, be specifically related to a kind of method of synthetic aviptadil.
Background technology
Aviptadil (Aviptadil) is a kind of nerve polypeptide, and except the nerve conduction effect or the vasoactive peptide agonists, its peptide order is as follows:
NH 2-His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-COONH 2
Press the patient to use aviptadil to pulmonary artery high (PH), find that it can cause of short duration highly selective lung vasodilation, often rich work output and mixed venous oxygen saturation increase, and can improve the oxygenate with pulmonary disorder patient.The III phase clinical experiment that aviptadil is used for the treatment of pulmonary hypertension enters the preparatory stage, and the II clinical trial phase that is used for sarcoidosis of lung is completed, is used for the II clinical trial phase well afoot of acute respiratory distress syndrome and idiopathic pulmonary fibrosis.
At present the conventional synthetic method of aviptadil is coupling method one by one, other synthetic methods also do not have report, more common about the report of its formulation and application, be aviptadil liquid pharmaceutical formulation and for the preparation of the use patent of the medicine of the diseases such as treatment pulmonary hypertension, acute respiratory distress syndrome as patent CN101247794; US7468353 is the use patent in the treatment interstitial lung infections.Yet with coupling method is in the process of preparation aviptadil one by one, synthesis cycle is long, and impurity is more, and yield and purity can't reach higher level, particularly synthetic His 1And Ser 2The time, coupling method very easily produces racemization impurity one by one, and impurity is difficult to remove by purification process, causes the reduction of aviptadil product purity and impurity D-His 1-aviptadil and D-Ser 2-aviptadil too high levels affects product quality.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of method of synthetic aviptadil, make the method for the invention can improve its purity, reduce impurity D-His 1-aviptadil and D-Ser 2-aviptadil content.
For achieving the above object, the invention provides following technical scheme:
A kind of method of synthetic aviptadil comprises the following steps:
Step 1, solid phase synthesis have protecting group and hold coupling that the polypeptide fragment resin I of resin is arranged at C in coupling on aminoacid sequence Lys side chain shown in SEQ ID NO:1, on the Tyr side chain, on the Asn side chain, on the Ser side chain;
Solid phase synthesis coupling on the end of aminoacid sequence N shown in SEQ ID NO:2, Tyr side chain, on the Thr side chain, on the Arg side chain, on the Lys side chain, on the Gln side chain has protecting group and the polypeptide fragment resin II of resin is arranged in the coupling of C end;
Solid phase synthesis coupling on the end of aminoacid sequence N shown in SEQ ID NO:3, Asp side chain, on the Thr side chain, on the Asn side chain has protecting group and the polypeptide fragment resin III of resin is arranged in the coupling of C end, then the N end protecting group and Boc-His (3-Bum)-Ser (Psi (Me, Me) the pro)-OH coupling that remove the polypeptide fragment resin III obtain polypeptide fragment resin IV;
Step 2, add lysate to remove the resin of polypeptide fragment resin II and polypeptide fragment resin IV, correspondence obtains polypeptide fragment II and polypeptide fragment IV, then the C section of polypeptide fragment II and the N of polypeptide fragment resin I are held coupling, follow the N section coupling with C section with the polypeptide fragment II that removes N end protecting group of polypeptide fragment IV, obtain the aviptadil resin;
Step 3, add lysate to remove resin and all protecting groups in the aviptadil resin, obtain the aviptadil crude product, obtain the aviptadil sterling after the RP-HPLC purifying.
Wherein, as preferably, PyBOP/HOBt/DIPEA three reagent coupling systems, HOAt/DIPCDI double reagent coupling system, HOBt/DIPCDI double reagent coupling system, HATU/HOAt/DIPEA three reagent coupling systems, TBTU/HOBt/DIPEA three reagent coupling systems or HBTU/HOBt/DIPEA three reagent coupling system couplings are adopted in the described coupling of step 2.Mol ratio between each coupling reagent of the coupling system that step 2 adopts is preferably:
PyBOP:HOBt:DIPEA is 1:1.2:2, and HATU:HOAt:DIPEA is 1:1.2:2, and HBTU:HOBt:DIPEA is 1:1.2:2, and TBTU:HOBt:DIPEA is 1:1.2:2, and HOAt:DIPCDI is 1.2:1.2, and HOBt:DIPCDI is 1.2:1.2;
As preferably, the described solid-phase synthetic peptide fragment of step 1 resin I is:
Steps A 1, Fmoc-Asn(Trt)-OH carries out linked reaction with resin and obtains Fmoc-Asn(Trt under HOBt/DIPCDI double reagent coupling system or organic bases DIPEA effect)-resin;
Steps A 2, remove the Fmoc protecting group and obtain H-Asn(Trt)-resin, Fmoc-Leu-OH under the effect of HOBt/DIPCDI double reagent coupling system with H-Asn(Trt)-resin carries out linked reaction and obtains Fmoc-Leu-Asn(Trt)-resin;
steps A 3, hold the order of N end according to aminoacid sequence C shown in SEQ ID NO:1, successively one by one with Fmoc-Ile-OH, Fmoc-Ser (Trt)-OH, Fmoc-Asn (Trt)-OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Val-OH carries out amino acid according to steps A 2 coupling modes and extends coupling, remove at last N end Fmoc protecting group and obtain H-Val-Lys (Boc)-Lys (Boc)-Tyr (tBu)-Leu-Asn (Trt)-Ser (Trt)-Ile-Leu-Asn (Trt)-resin, it is polypeptide fragment resin I,
As preferably, the described solid-phase synthetic peptide fragment of step 1 resin II is:
Step B1, Fmoc-Ala-OH carry out linked reaction with resin and obtain the Fmoc-Ala-resin under the DIPEA effect;
Step B2, remove the Fmoc protecting group and obtain the H-Ala-resin, Fmoc-Met-OH carries out linked reaction with the H-Ala-resin and obtains the Fmoc-Met-Ala-resin under the effect of HOBt/DIPCDI double reagent coupling system;
step B3, hold the order of N end according to aminoacid sequence C shown in SEQ ID NO:2, successively one by one with Fmoc-Gln (Trt)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Leu-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Thr (tBu)-OH, Fmoc-Tyr (tBu)-OH carries out amino acid according to step B2 coupling mode and extends coupling, obtain Fmoc-Tyr (tBu)-Thr (tBu)-Arg (Pbf)-Leu-Arg (Pbf)-Lys (Boc)-Gln (Trt)-Met-Ala-resin, it is polypeptide fragment resin II, wherein, described Fmoc is amino acid N end protecting group, and described tBu, Trt, Boc, Pbf are the amino acid side chain protecting group.
As preferably, the described solid-phase synthetic peptide fragment of step 1 resin III is:
Step C1, Fmoc-Asn (Trt)-OH carries out linked reaction with resin and obtains Fmoc-Asn (Trt)-resin under the DIPEA effect;
Step C2, remove the Fmoc protecting group and obtain H-Asn (Trt)-resin, Fmoc-Asp (OtBu)-OH carries out linked reaction with H-Asn (Trt)-resin and obtains Fmoc-Asp (OtBu)-Asn (Trt)-resin under the effect of HOBt/DIPCDI double reagent coupling system;
Step C3, the order of holding the N end according to aminoacid sequence C shown in SEQ ID NO:3, one by one Fmoc-Thr (tBu)-OH, Fmoc-Phe-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Asp (OtBu)-OH are carried out amino acid according to step C2 coupling mode successively and extend coupling, obtain Fmoc-Asp (OtBu)-Ala-Val-Phe-Thr (tBu)-Asp (OtBu)-Asn (Trt)-resin, i.e. polypeptide fragment resin III;
step C4, DBLK removes the N end Fmoc protecting group of polypeptide fragment resin III, with Boc-His (3-Bum)-Ser (Psi (Me, Me) pro)-OH is at PyBOP/HOBt/DIPEA three reagent coupling systems, HOAt/DIPCDI double reagent coupling system, HOBt/DIPCDI double reagent coupling system, HATU/HOAt/DIPEA three reagent coupling systems, TBTU/HOBt/DIPEA three reagent coupling systems, or under HBTU/HOBt/DIPEA three reagent coupling system effects, coupling obtains Boc-His (3-Bum)-Ser (Psi (Me, Me) pro)-Asp (OtBu)-Ala-Val-Phe-Thr (tBu)-Asp (OtBu)-Asn (Trt)-resin, it is polypeptide fragment resin IV, wherein, described Fmoc, Boc are amino acid N end protecting group, and described tBu, OtBu, Trt are the amino acid side chain protecting group.As preferably, the mol ratio of described Boc-His (3-Bum)-Ser (Psi (Me, Me) pro)-OH and HOBt, HOAt, HBTU, HATU, TBTU, PyBOP, DIPCDI, DIPEA is 1:1.2:1.2:1:1:1:1:1.2:2.
In technical solution of the present invention and optimal technical scheme, described resin preferably adopts substitution degree to be CTC resin, Rink Amide resin, Rink Amide-AM resin or the RinkAmide-MBHA resin of 0.2-1.0mmol/g.As preferably, when solid-phase synthetic peptide fragment resin I, resin is preferably Rink Amide resin, Rink Amide-AM resin or Rink Amide-MBHA resin; When solid-phase synthetic peptide fragment resin II, III, resin is preferably the CTC resin.In three preferred synthesis steps of polypeptide fragment resin of step 1, except the protecting group that the present invention limits, also can adopt other suitable protecting groups that amino acid is protected.
In technical solution of the present invention and optimal technical scheme, all one or both in DCM, NMP, DMF, the DMSO are as solvent in described coupling, and wherein, during as solvent, the volume ratio of two kinds of solvents is preferably 1:1 when two kinds in DCM, NMP, DMF, DMSO.In addition, the temperature of reaction of all linked reactions of the present invention all is preferably 0-30 ℃.。In three preferred synthesis steps of polypeptide fragment resin of step 1, the mol ratio of described each protected amino acid and HOBt, DIPCDI, DIPEA is preferably 1:1.2:1.2:2.
As preferably; the described polypeptide fragment II of step 3, polypeptide fragment IV and the mol ratio that removes the polypeptide fragment resin I of N segment protect base are 2-4:2-4:1; the described lysate of step 3 is that volume ratio TFE:DCM is the mixed pyrolysis liquid of 1:4, and the described lysate of step 4 is that volume ratio TFA:PhSMe:TIS:EDT:PhOH is the mixed pyrolysis liquid cracking of 80-90:0-4:1-4:2-4:1-5.
In the method for the invention, at first according to aviptadil peptide order synthetic 1-9,10-18 and 19-28 fragment, and then these 3 polypeptide fragment couplings are obtained aviptadil, wherein first to synthesize when synthetic 1-9 fragment, the 3-9 fragment, again with special synthesis material Boc-His (3-Bum)-Ser (Psi (Me, Me) pro)-OH synthesizes the 1-9 fragment, solved in existing synthetic method the improper racemization impurity that causes due to synthesis material and coupling method, finally cause the aviptadil purity drop, D-His 1-aviptadil and D-Ser 2The problem that-aviptadil content is higher.Hold the amino-acid sequence numbering of C end with aviptadil main chain N, as shown in the formula:
NH 2-His 1-Ser 2-Asp 3-Ala 4-Val 5-Phe 6-Thr 7-Asp 8-Asn 9-Tyr 10-Thr 11-Arg 12-Leu 13-Arg 14-Lys 15-Gln 16-Met 17-Ala 18-Val 19-Lys 20-Lys 21-Tyr 22-Leu 23-Asn 24-Ser 25-Ile 26-Leu 27-Asn 28-COONH 2
Aminoacid sequence shown in SEQ ID NO:1 is the peptide sequence of numbering 19-28 in following formula.The present invention's polypeptide fragment resin I of solid phase synthesis in step 1 is on aminoacid sequence basis shown in SEQ ID NO:1, protecting group is arranged and in the coupling of C end, resin is arranged in coupling on its Lys side chain, on the Tyr side chain, on the Asn side chain, on the Ser side chain; Aminoacid sequence shown in SEQ ID NO:2 is the peptide sequence of numbering 10-18 in following formula, polypeptide fragment resin II is on aminoacid sequence basis shown in SEQ ID NO:2, and coupling has protecting group and in the coupling of C end, resin arranged on its N end, Tyr side chain, on the Thr side chain, on the Arg side chain, on the Lys side chain, on the Gln side chain; Aminoacid sequence shown in SEQ ID NO:3 is the peptide sequence of numbering 3-9 in following formula, the polypeptide fragment resin III is on aminoacid sequence basis shown in SEQ ID NO:3, and coupling has protecting group and in the coupling of C end, resin arranged on its N end, Asp side chain, on the Thr side chain, on the Asn side chain; Abovely can synthesize by the protected amino acid synthesis material in the art.
Protecting group of the present invention be in amino acid synthetic field in order to protected amino acid main chain and side chain on amino, carboxyl, sulfydryl etc. disturb the blocking group of the group that synthesizes; prevent that amino, carboxyl etc. from reacting, and generates impurity in preparation target product process.In the art, the group, side-chain structure and the how coupling protecting group that need protection for amino acid side chain are known to the skilled person.In the present invention, to be associated with the amino acid representation of protecting group be also this area representation commonly used to antithesis; be well known to those skilled in the art; as Fmoc-Asp(OtBu)-OH; Fmoc is amino acid N end protecting group; OtBu in bracket is the Asp Side chain protective group, and other protected amino acid synthesis materials of the present invention all can illustrate with reference to this.
In the preferred version of step 1 solid-phase synthetic peptide fragment resin, described extension coupling refers to after first amino acid and resin coupling, and remaining amino acid carries out coupling with the amino acid generation condensation reaction of previous coupling (condensation reaction of the amino and carboxyl of main chain) one by one according to the order of sequence separately.In extending coupling; because each amino acid N end has protecting group, therefore need to first remove the coupling again of N end Fmoc protecting group, this is common practise for a person skilled in the art; the present invention is preferably with the i.e. 20% piperidines/DMF of DBLK(, volume ratio) remove N end protecting group.Due to amino acid and resin coupling constantly being arranged; the polypeptide fragment resin that is synthesized is constantly to change; as preferably, each treat the protected amino acid synthetic materials of coupling with before the mol ratio of synthetic polypeptide fragment resin be 2-3:1, this preferred proportion is applicable in all schemes of the present invention.
As preferably, after step 2 obtains polypeptide fragment II and polypeptide fragment IV, also comprise the refining step to polypeptide fragment II and polypeptide fragment IV:
Adopt polar solvent dissolving polypeptide fragment II or polypeptide fragment IV, then be added drop-wise in inert solvent and stir, wash with inert solvent after filtering, namely complete refining step after drying, wherein, described polar solvent is methyl alcohol, ethyl acetate or methylene dichloride, and described inert solvent is ether, normal hexane, sherwood oil or methyl tertiary butyl ether.
In the method for the invention step 3, as preferred version, described RP-HPLC purifying is specially:
Be after 10% acetonitrile water ultrasonic dissolution with aviptadil crude product percent by volume, adopt the RP-HPLC system, wavelength 214nm, chromatographic column is the anti-phase C18 post of 50 * 250mm, conventional 0.1%TFA/ acetonitrile moving phase purifying is collected purpose peak cut and is obtained the aviptadil sterling.
In clinical application, the less stable of aviptadil own need to exist with aviptadil acetate form, therefore the present invention also comprises, the aviptadil sterling is turned the salt step with the RP-HPLC system, is specially:
Aviptadil sterling solution is adopted the RP-HPLC system, and chromatographic column is the anti-phase C18 post of 50 * 250mm, and 0.1% acetum/acetonitrile moving phase turns salt, collects purpose peak cut, and rotary evaporation is concentrated, and freeze-drying obtains aviptadil acetate.
The aviptadil synthetic by the method for the invention detects through RP-HPLC, and purity is more than 80%, impurity D-His 1-aviptadil is in 1.5% left and right, impurity D-Ser 2-aviptadil is in 3% left and right, and the aviptadil that adopts conventional raw material and synthetic method to synthesize, purity is 75%, impurity D-His 1-aviptadil is in 6% left and right, impurity D-Ser 2-aviptadil is in 7% left and right.
By above technical scheme as can be known, at first the present invention adopts suitable raw material according to aviptadil peptide order synthetic 1-9,10-18 and 19-28 fragment, and then these 3 polypeptide fragment couplings are obtained aviptadil, solved in existing synthetic method the improper racemization foreign matter content that causes due to synthesis material and coupling method higher, the problem that aviptadil purity is lower.
Description of drawings
Figure 1 shows that the aviptadil crude product RP-HPLC peak figure that synthetic method of the present invention is synthetic;
Figure 2 shows that with the synthetic aviptadil crude product RP-HPLC peak figure of the coupling method one by one of routine.
Embodiment
The invention discloses a kind of method of synthetic aviptadil, those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is, all similarly replace and change apparent to those skilled in the art, and they all are deemed to be included in the present invention.Method of the present invention is described by preferred embodiment, the related personnel obviously can be within not breaking away from content of the present invention, spirit and scope to compound as herein described with the preparation method changes or suitably change and combination, realize and use the technology of the present invention.
In the specific embodiment of the invention; all couplings all can be by commercially available acquisition by the amino acid of protecting group; protected amino acid in the present invention is available from the biochemical company limited of gill; resin used available from Tianjin Nankai with become company limited, the Chinese implication that in application documents, english abbreviation used is corresponding sees Table 1.
The lexical or textual analysis of table 1 english abbreviation
Figure BDA00002967049600071
Figure BDA00002967049600081
Below in conjunction with embodiment, further set forth the present invention.
Embodiment 1: polypeptide fragment resin I synthetic
Taking substitution degree is the Rink Amide resin 100g(20mmol of 0.2mmol/g); join in the solid state reaction post; with DMF washing 2 times; with DMF swelling resin after 30 minutes; add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes are after reaction finishes; with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin has color.Take 23.87g Fmoc-Asn (Trt)-OH(40mmol), HOBt5.94g(44mmol) use 80mlDMF/DCM (1:1, V:V) dissolving, add 6.85ml DIPCDI(44mmol under ice-water bath) activation 3 minutes after, add in the above-mentioned reaction column that resin is housed, reacted 2 hours, reaction end adopts triketohydrindene hydrate to detect to judge (if the resin water white transparency reacts completely; If developing the color, resin continues reflection 1 hour.Lower same), after reaction finishes, use DMF washing resin 3 times.
Add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes, after reaction finished, with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin had color.Adopt identical method coupling Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Ser (Trt)-OH, Fmoc-Asn (Trt)-OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Val-OH.
After finishing, the Fmoc-Val-OH coupling uses DMF washing resin 3 times.Add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes, after reaction finished, with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin had color.Obtain removing the polypeptide fragment resin I:H-Val-Lys (Boc) of N end Fmoc protecting group-Lys (Boc)-Tyr (tBu)-Leu-Asn (Trt)-Ser (Trt)-Ile-Leu-Asn (Trt)-Rink Amide resin, stand-by.
Embodiment 2: polypeptide fragment resin I synthetic
Taking substitution degree is the Rink Amide resin 33.3g(20mmol of 0.6mmol/g); join in the solid state reaction post; with DMF washing 2 times; with DMF swelling resin after 30 minutes; add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes are after reaction finishes; with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin has color.Take 23.87g Fmoc-Asn (Trt)-OH(40mmol), HOBt5.94g(44mmol) use 80ml DMF/DCM (1:1, V:V) dissolving, add 6.85mlDIPCDI(44mmol under ice-water bath) activation 3 minutes after, add in the above-mentioned reaction column that resin is housed, reacted 2 hours, reaction end adopts triketohydrindene hydrate to detect to judge (if the resin water white transparency reacts completely; If developing the color, resin continues reflection 1 hour.Lower same), after reaction finishes, use DMF washing resin 3 times.
Add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes, after reaction finished, with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin had color.Adopt identical method coupling Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Ser (Trt)-OH, Fmoc-Asn (Trt)-OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Val-OH.
After finishing, the Fmoc-Val-OH coupling uses DMF washing resin 3 times.Add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes, after reaction finished, with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin had color.Obtain removing the polypeptide fragment resin I:H-Val-Lys (Boc) of N end Fmoc protecting group-Lys (Boc)-Tyr (tBu)-Leu-Asn (Trt)-Ser (Trt)-Ile-Leu-Asn (Trt)-Rink Amide resin, stand-by.
Embodiment 3: polypeptide fragment resin I synthetic
Taking substitution degree is the Rink Amide resin 20g(20mmol of 1.0mmol/g); join in the solid state reaction post; with DMF washing 2 times; with DMF swelling resin after 30 minutes; add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes are after reaction finishes; with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin has color.Take 23.87g Fmoc-Asn (Trt)-OH(40mmol), HOBt5.94g(44mmol) use 80ml DMF/DCM (1:1, V:V) dissolving, add 6.85mlDIPCDI(44mmol under ice-water bath) activation 3 minutes after, add in the above-mentioned reaction column that resin is housed, reacted 2 hours, reaction end adopts triketohydrindene hydrate to detect to judge (if the resin water white transparency reacts completely; If developing the color, resin continues reflection 1 hour.Lower same), after reaction finishes, use DMF washing resin 3 times.
Add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes, after reaction finished, with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin had color.Adopt identical method coupling Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Ser (Trt)-OH, Fmoc-Asn (Trt)-OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Val-OH.
After finishing, the Fmoc-Val-OH coupling uses DMF washing resin 3 times.Add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes, after reaction finished, with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin had color.Obtain removing the polypeptide fragment resin I:H-Val-Lys (Boc) of N end Fmoc protecting group-Lys (Boc)-Tyr (tBu)-Leu-Asn (Trt)-Ser (Trt)-Ile-Leu-Asn (Trt)-Rink Amide resin, stand-by.
Embodiment 4: polypeptide fragment resin I synthetic
Taking substitution degree is the Rink Amide-AM resin 33.3g(20mmol of 0.6mmol/g); join in the solid state reaction post; with DMF washing 2 times; with DMF swelling resin after 30 minutes; add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes are after reaction finishes; with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin has color.Take 23.87g Fmoc-Asn (Trt)-OH(40mmol), HOBt5.94g(44mmol) use 80ml DMF/DCM (1:1, V:V) dissolving, add 6.85ml DIPCDI(44mmol under ice-water bath) activation 3 minutes after, add in the above-mentioned reaction column that resin is housed, reacted 2 hours, reaction end adopts triketohydrindene hydrate to detect to judge (if the resin water white transparency reacts completely; If developing the color, resin continues reflection 1 hour.Lower same), after reaction finishes, use DMF washing resin 3 times.
Add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes, after reaction finished, with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin had color.Adopt identical method coupling Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Ser (Trt)-OH, Fmoc-Asn (Trt)-OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Val-OH.
After finishing, the Fmoc-Val-OH coupling uses DMF washing resin 3 times.Add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes, after reaction finished, with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin had color.Obtain removing the polypeptide fragment resin I:H-Val-Lys (Boc) of N end Fmoc protecting group-Lys (Boc)-Tyr (tBu)-Leu-Asn (Trt)-Ser (Trt)-Ile-Leu-Asn (Trt)-Rink Amide-AM resin, stand-by.
Embodiment 5: polypeptide fragment resin I synthetic
Taking substitution degree is the Rink Amide-AM resin 33.3g(20mmol of 0.6mmol/g); join in the solid state reaction post; with DMF washing 2 times; with DMF swelling resin after 30 minutes; add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes are after reaction finishes; with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin has color.Take 23.87g Fmoc-Asn (Trt)-OH(40mmol), HOBt5.94g(44mmol) use 80ml DMF/DCM (1:1, V:V) dissolving, add 6.85ml DIPCDI(44mmol under ice-water bath) activation 3 minutes after, add in the above-mentioned reaction column that resin is housed, reacted 2 hours, reaction end adopts triketohydrindene hydrate to detect to judge (if the resin water white transparency reacts completely; If developing the color, resin continues reflection 1 hour.Lower same), after reaction finishes, use DMF washing resin 3 times.
Add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes, after reaction finished, with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin had color.Adopt identical method coupling Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Ser (Trt)-OH, Fmoc-Asn (Trt)-OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Val-OH.
After finishing, the Fmoc-Val-OH coupling uses DMF washing resin 3 times.Add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes, after reaction finished, with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin had color.Obtain removing the polypeptide fragment resin I:H-Val-Lys (Boc) of N end Fmoc protecting group-Lys (Boc)-Tyr (tBu)-Leu-Asn (Trt)-Ser (Trt)-Ile-Leu-Asn (Trt)-Rink Amide-MBHA resin, stand-by.
Embodiment 6: polypeptide fragment resin I synthetic
Taking substitution degree is the Rink Amide-AM resin 40g(20mmol of 0.5mmol/g); join in the solid state reaction post; with DMF washing 2 times; with DMF swelling resin after 30 minutes; add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes are after reaction finishes; with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin has color.Take 23.87g Fmoc-Asn (Trt)-OH(40mmol), HOBt5.94g(44mmol) use 80ml DMF/DCM (1:1, V:V) dissolving, add 6.85mlDIPCDI(44mmol under ice-water bath) activation 3 minutes after, add in the above-mentioned reaction column that resin is housed, reacted 2 hours, reaction end adopts triketohydrindene hydrate to detect to judge (if the resin water white transparency reacts completely; If developing the color, resin continues reflection 1 hour.Lower same), after reaction finishes, use DMF washing resin 3 times.
Add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes, after reaction finished, with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin had color.Adopt identical method coupling Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Ser (Trt)-OH, Fmoc-Asn (Trt)-OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Val-OH.
After finishing, the Fmoc-Val-OH coupling uses DMF washing resin 3 times.Add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes, after reaction finished, with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin had color.Obtain removing the polypeptide fragment resin I:H-Val-Lys (Boc) of N end Fmoc protecting group-Lys (Boc)-Tyr (tBu)-Leu-Asn (Trt)-Ser (Trt)-Ile-Leu-Asn (Trt)-Rink Amide-AM resin, stand-by.
Embodiment 7: polypeptide fragment resin II synthetic
Taking substitution degree is the 2-CTC resin 40g(20mmol of 0.5mmol/g), join in the solid state reaction post, with DMF washing 2 times, with DMF swelling resin after 30 minutes, get 12.4g Fmoc-Ala-OH(40mmol) with 70mlDMF dissolving, add 14.1ml DIPEA(80mmol under ice-water bath) activation is after 3 minutes, add in the above-mentioned reaction column that resin is housed, react after 2 hours, add 15ml anhydrous methanol sealing 0.5 hour.With DMF washing 3 times, DCM washes 3 times.
Add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes, after reaction finished, with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin had color.Take Fmoc-Met-OH15.5g and 5.94g HOBt, with the 90mlDMF dissolving, add DIPCDI6.85ml activation 5 minutes under ice-water bath, join in reaction column, room temperature reaction 2 hours, reaction end adopt triketohydrindene hydrate to detect to judge (if the resin water white transparency reacts completely; If developing the color, resin continues reflection 1 hour.Lower same), after reaction finishes, use DMF washing resin 3 times.
Add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes, after reaction finished, with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin had color.Adopt identical method coupling Fmoc-Gln (Trt)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Leu-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Thr (tBu)-OH, Fmoc-Tyr (tBu)-OH.Reaction finishes, DMF washes 3 times, DCM washes 3 times, methyl alcohol shrinks, drain, obtain polypeptide fragment resin II: Fmoc-Tyr (tBu)-Thr (tBu)-Arg (Pbf)-Leu-Arg (Pbf)-Lys (Boc)-Gln (Trt)-Met-Ala-2-C TC resin 79.8g.
with the polypeptide fragment resin II 79.8g that obtains, add pre-configured lysate TFE:DCM=1:4(V:V) 800ml, room temperature cracking 2.5 hours, filter, 30ml DCM washing, merging filtrate, be evaporated to 80ml, be added drop-wise to the 800ml anhydrous diethyl ether and separate out white solid, centrifugally drain that to obtain white solid (be polypeptide fragment II Fmoc-Tyr (tBu)-Thr (tBu)-Arg (Pbf)-Leu-Arg (Pbf)-Lys (Boc)-Gln (Trt)-Met-Ala-OH) 39.1g, yield 85.1%, HPLC purity is 88.5%, standby.
Embodiment 8: polypeptide fragment resin III and polypeptide fragment resin IV synthetic
Taking substitution degree is the 2-CTC resin 40g(20mmol of 0.5mmol/g), join in the solid state reaction post, with DMF washing 2 times, with DMF swelling resin after 30 minutes, get 23.87g Fmoc-Asn (Trt)-OH(40mmol) with 70ml DMF dissolving, add 14.1ml DIPEA(80mmol under ice-water bath) activation is after 3 minutes, adds in the above-mentioned reaction column that resin is housed, react after 2 hours, add 15ml anhydrous methanol sealing 0.5 hour.With DMF washing 3 times, DCM washes 3 times.
Add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes, after reaction finished, with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin had color.Take Fmoc-Asp (OtBu)-OH17.1g and 5.94g HOBt, dissolve with 90mlDMF, add DIPCDI6.85ml activation 5 minutes under ice-water bath, join in reaction column, room temperature reaction 2 hours, reaction end adopts triketohydrindene hydrate to detect to judge (if the resin water white transparency reacts completely; If developing the color, resin continues reflection 1 hour.Lower same), after reaction finishes, use DMF washing resin 3 times.
Add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes, after reaction finished, with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin had color.Adopt identical method coupling Fmoc-Thr (tBu)-OH, Fmoc-Phe-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Asp (OtBu)-OH obtains polypeptide fragment resin III: Fmoc-Asp (OtBu)-Ala-Val-Phe-Thr (tBu)-Asp (OtBu)-Asn (Trt)-2-CTC.
Add the solution 100ml of 20% piperidines/DMF(V:V) to the polypeptide fragment resin III, deprotection 5 and 10 minutes, after reaction finished, with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin had color.Take Boc-His (3-Bum)-Ser (Psi (Me, Me) pro)-OH28.1g, PyBOP31.2g and 8.9g HOBt, dissolve with 100mlDMF, add DIPEA20.9ml activation 5 minutes under ice-water bath, join in reaction column room temperature reaction 2 hours, reaction end adopts triketohydrindene hydrate to detect to judge (if the resin water white transparency reacts completely; If developing the color, resin continues reflection 1 hour.Lower same), after reaction finishes, with DMF washing resin 3 times, DCM washes 3 times, methyl alcohol shrinks, drain, obtain polypeptide fragment resin IV: Boc-His (3-Bum)-Ser (Psi (Me, Me) pro)-Asp (OtBu)-Ala-Val-Phe-Thr (tBu)-Asp (OtB u)-Asn (Trt)-2-CTC66.3g.
with the 66.3g polypeptide fragment resin IV that obtains, add pre-configured lysate TFE:DCM=1:4(V:V) 670ml, room temperature cracking 2.5 hours, filter, 30ml DCM washing, merging filtrate, be evaporated to 60ml, be added drop-wise to the 600ml anhydrous diethyl ether and separate out white solid, centrifugally drain that to obtain white solid (be polypeptide fragment IV Boc-His (3-Bum)-Ser (Psi (Me, Me) pro)-Asp (OtBu)-Ala-Val-Phe-Thr (tBu)-Asp (OtB u)-Asn (Trt)-OH) 26.1g, yield 80.4%.HPLC purity is 92.1%, and is standby
Embodiment 9: polypeptide fragment resin III and polypeptide fragment resin IV synthetic
Taking substitution degree is the 2-CTC resin 40g(20mmol of 0.5mmol/g), join in the solid state reaction post, with DMF washing 2 times, with DMF swelling resin after 30 minutes, get 23.87gFmoc-Asn (Trt)-OH(40mmol) with 70ml DMF dissolving, add 14.1ml DIPEA(80mmol under ice-water bath) activation is after 3 minutes, adds in the above-mentioned reaction column that resin is housed, react after 2 hours, add 15ml anhydrous methanol sealing 0.5 hour.With DMF washing 3 times, DCM washes 3 times.
Add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes, after reaction finished, with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin had color.Take Fmoc-Asp (OtBu)-OH17.1g, HBTU22.8g and 8.9g HOBt, dissolve with 100mlDMF, add DIPEA20.9ml activation 5 minutes under ice-water bath,, join in reaction column room temperature reaction 2 hours, reaction end adopts triketohydrindene hydrate to detect to judge (if the resin water white transparency reacts completely; If developing the color, resin continues reflection 1 hour.Lower same), after reaction finishes, use DMF washing resin 3 times.
Add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes, after reaction finished, with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin had color.Adopt identical method coupling Fmoc-Thr (tBu)-OH, Fmoc-Phe-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Asp (OtBu)-OH obtains polypeptide fragment resin III: Fmoc-Asp (OtBu)-Ala-Val-Phe-Thr (tBu)-Asp (OtBu)-Asn (Trt)-2-CTC.
Add the solution 100ml of 20% piperidines/DMF(V:V) to the polypeptide fragment resin III, deprotection 5 and 10 minutes, after reaction finished, with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin had color.Take Boc-His (3-Bum)-Ser (Psi (Me, Me) pro)-OH28.1g, HBTU22.8g and 8.9g HOBt, dissolve with 100mlDMF, add DIPEA20.9ml activation 5 minutes under ice-water bath, join in reaction column room temperature reaction 2 hours, reaction end adopts triketohydrindene hydrate to detect to judge (if the resin water white transparency reacts completely; If developing the color, resin continues reflection 1 hour.Lower same), after reaction finishes, with DMF washing resin 3 times, DCM washes 3 times, methyl alcohol shrinks, drain, obtain polypeptide fragment resin IV: Boc-His (3-Bum)-Ser (Psi (Me, Me) pro)-Asp (OtBu)-Ala-Val-Phe-Thr (tBu)-Asp (OtB u)-Asn (Trt)-CTC68.5g.
With the 68.5g polypeptide fragment resin IV that obtains, add pre-configured lysate TFE:DCM=1:4(V:V) 690ml, room temperature cracking 2.5 hours, filter, 30ml DCM washing, merging filtrate, be evaporated to 60ml, be added drop-wise to the 600ml anhydrous diethyl ether and separate out white solid, centrifugally drain that to obtain white solid (be polypeptide fragment IV Boc-His (3-Bum)-Ser (Psi (Me, Me) pro)-Asp (OtBu)-Ala-Val-Phe-Thr (tBu)-Asp (OtBu)-Asn (Trt)-OH) 28.2g, yield 86.1%.HPLC purity is 93.6%.
Embodiment 10: polypeptide fragment II refining
Embodiment 7 is obtained polypeptide fragment II 20 grams 40ml dissolve with methanol, slowly be added drop-wise in the 200ml ether, separate out white solid, stirring at room is 30 minutes again, filters, and filter cake washs with the 20ml anhydrous diethyl ether, the vacuum-drying of gained solid, white solid after being made with extra care (the polypeptide fragment II after refining) 16.6g, HPLC purity 95.0%, standby.
Embodiment 11: polypeptide fragment II refining
Embodiment 7 is obtained polypeptide fragment II 20 grams to be dissolved with the 30ml tetrahydrofuran (THF), slowly be added drop-wise in the 200ml normal hexane, separate out white solid, stirring at room is 30 minutes again, filters, and filter cake washs with the 20ml normal hexane, the vacuum-drying of gained solid, white solid after being made with extra care (the polypeptide fragment II after refining) 18.2g, HPLC purity 96.9%, standby.
Embodiment 12: polypeptide fragment IV's is refining
Embodiment 9 is obtained polypeptide fragment IV 20 grams 30ml acetic acid ethyl dissolution, slowly be added drop-wise in the 300ml sherwood oil, separate out white solid, then stirring at room 30 minutes, standing 2 hours, filter, filter cake 20ml petroleum ether, the vacuum-drying of gained solid, the white solid after being made with extra care (the polypeptide fragment IV after refining) 18.6g, HPLC purity 98.5%, standby.
Embodiment 13: polypeptide fragment IV's is refining
Embodiment 9 is obtained polypeptide fragment IV 20 grams 30ml acetone solution, slowly be added drop-wise in 200ml water, separate out white solid, then stirring at room 30 minutes, standing 2 hours, filter, filter cake is used 20ml water successively, the washing of 40ml anhydrous diethyl ether, the vacuum-drying of gained solid, white solid after being made with extra care (the polypeptide fragment IV after refining) 18.4g, HPLC purity 97.1%, standby.
Embodiment 14: the preparation of aviptadil resin
take 9.36g that embodiment 11 the obtains polypeptide fragment II after refining, HOAt0.61g, with 50mlDMF/DCM (1:1, V:V) dissolving, add 0.68ml DIPCDI activation 4 minutes under ice-water bath, join in the polypeptide fragment resin I:H-Val-Lys (Boc) that removes N end protecting group that embodiment 2 obtains-Lys (Boc)-Tyr (tBu)-Leu-Asn (Trt)-Ser (Trt)-Ile-Leu-Asn (Trt)-Rink Amide resin (2mmol), room temperature reaction 3 hours, reaction end adopts triketohydrindene hydrate to detect to judge (if resin water white transparency, react completely, if developing the color, resin continues reflection 1 hour.Lower with), after reaction finishes, with DMF washing resin 3 times, add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes, after reaction finished, with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin had color.
Take 6.56g that embodiment 12 the obtains polypeptide fragment IV after refining, HATU1.52g and HOAt0.61g, dissolve with 50ml DCM, add 0.68ml DIPCDI activation 4 minutes under ice-water bath, join in above-mentioned reaction column, room temperature reaction 3 hours, reaction end adopt triketohydrindene hydrate to detect to judge (if the resin water white transparency reacts completely; If developing the color, resin continues reflection 1 hour.Lower same), after reaction finishes, DCM washing 3 times, methyl alcohol shrinks, and drains, and obtains aviptadil resin 12.8g, resin rate of body weight gain 82.3%.
Embodiment 15: the preparation of aviptadil resin
take 9.36g that embodiment 11 the obtains polypeptide fragment II after refining, HATU1.52g and HOAt0.61g, with 50ml DMF/DCM (1:1, V:V) dissolving, add 1.4ml DIPEA activation 4 minutes under ice-water bath, join in the polypeptide fragment resin I:H-Val-Lys (Boc) that removes N end protecting group that embodiment 2 obtains-Lys (Boc)-Tyr (tBu)-Leu-Asn (Trt)-Ser (Trt)-Ile-Leu-Asn (Trt)-Rink Amide peptide resin (2mmol), room temperature reaction 3 hours, reaction end adopts triketohydrindene hydrate to detect to judge (if resin water white transparency, react completely, if developing the color, resin continues reflection 1 hour.Lower with), after reaction finishes, with DMF washing resin 3 times, add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes, after reaction finished, with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin had color.
Take 6.56g that embodiment 12 the obtains polypeptide fragment IV after refining, HATU1.52g and HOAt0.61g, dissolve with 50ml DCM, add 1.4ml DIPEA activation 4 minutes under ice-water bath, join in above-mentioned reaction column, room temperature reaction 3 hours, reaction end adopt triketohydrindene hydrate to detect to judge (if the resin water white transparency reacts completely; If developing the color, resin continues reflection 1 hour.Lower same), after reaction finishes, DCM washing 3 times, methyl alcohol shrinks, and drains, and obtains aviptadil resin 14.9g, resin rate of body weight gain 95.5%.
Embodiment 16: the preparation of aviptadil resin
take 9.36g that embodiment 11 the obtains polypeptide fragment II after refining, PyBOP2.1g and HOBt0.61g, dissolve with 50ml NMP, add 1.4ml DIPEA activation 4 minutes under ice-water bath, join in the polypeptide fragment resin I:H-Val-Lys (Boc) that removes N end protecting group that embodiment 2 obtains-Lys (Boc)-Tyr (tBu)-Leu-Asn (Trt)-Ser (Trt)-Ile-Leu-Asn (Trt)-Rink Amide peptide resin (2mmol), room temperature reaction 3 hours, reaction end adopts triketohydrindene hydrate to detect to judge (if resin water white transparency, react completely, if developing the color, resin continues reflection 1 hour.Lower with), after reaction finishes, with DMF washing resin 3 times, add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes, after reaction finished, with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin had color.
Take 6.56g that embodiment 12 the obtains polypeptide fragment IV after refining, PyBOP2.1g and HOBt0.61g, dissolve with 50ml NMP, add 1.4ml DIPEA activation 4 minutes under ice-water bath, join in above-mentioned reaction column, room temperature reaction 3 hours, reaction end adopt triketohydrindene hydrate to detect to judge (if the resin water white transparency reacts completely; If developing the color, resin continues reflection 1 hour.Lower same), after reaction finishes, DCM washing 3 times, methyl alcohol shrinks, and drains, and obtains aviptadil resin 14.4g, resin rate of body weight gain 92.1%.
Embodiment 17: the preparation of aviptadil resin
take 9.36g that embodiment 11 the obtains polypeptide fragment II after refining, TBTU1.3g and HOBt0.61g, dissolve with 50ml DMF, add 1.4ml DIPEA activation 4 minutes under ice-water bath, join in the polypeptide fragment resin I:H-Val-Lys (Boc) that removes N end protecting group that embodiment 2 obtains-Lys (Boc)-Tyr (tBu)-Leu-Asn (Trt)-Ser (Trt)-Ile-Leu-Asn (Trt)-Rink Amide peptide resin (2mmol), room temperature reaction 3 hours, reaction end adopts triketohydrindene hydrate to detect to judge (if resin water white transparency, react completely, if developing the color, resin continues reflection 1 hour.Lower with), after reaction finishes, with DMF washing resin 3 times, add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes, after reaction finished, with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin had color.
Take 6.56g that embodiment 12 the obtains polypeptide fragment IV after refining, TBTU1.3g and HOBt0.61g, dissolve with 50ml DMSO, add 1.4ml DIPEA activation 4 minutes under ice-water bath, join in above-mentioned reaction column, room temperature reaction 3 hours, reaction end adopt triketohydrindene hydrate to detect to judge (if the resin water white transparency reacts completely; If developing the color, resin continues reflection 1 hour.Lower same), after reaction finishes, DCM washing 3 times, methyl alcohol shrinks, and drains, and obtains aviptadil resin 12.5g, resin rate of body weight gain 80.3%.
Embodiment 18: the preparation of aviptadil resin
take 9.36g that embodiment 11 the obtains polypeptide fragment II after refining, HBTU1.5g and HOBt0.61g, dissolve with 50ml DMF, add 1.4ml DIPEA activation 4 minutes under ice-water bath, join in the polypeptide fragment resin I:H-Val-Lys (Boc) that removes N end protecting group that embodiment 2 obtains-Lys (Boc)-Tyr (tBu)-Leu-Asn (Trt)-Ser (Trt)-Ile-Leu-Asn (Trt)-Rink Amide peptide resin (2mmol), room temperature reaction 3 hours, reaction end adopts triketohydrindene hydrate to detect to judge (if resin water white transparency, react completely, if developing the color, resin continues reflection 1 hour.Lower with), after reaction finishes, with DMF washing resin 3 times, add the solution 100ml of 20% piperidines/DMF(V:V), deprotection 5 and 10 minutes, after reaction finished, with DMF washing resin 6 times, adopting triketohydrindene hydrate to detect resin had color.
Take 6.56g that embodiment 12 the obtains polypeptide fragment IV after refining, HBTU1.5g and HOBt0.61g, with 50ml DMSO/DCM (1:1, V:V) dissolving, add 1.4ml DIPEA activation 4 minutes under ice-water bath, join in above-mentioned reaction column room temperature reaction 3 hours, reaction end adopts triketohydrindene hydrate to detect to judge (if the resin water white transparency reacts completely; If developing the color, resin continues reflection 1 hour.Lower same), after reaction finishes, DCM washing 3 times, methyl alcohol shrinks, and drains, and obtains aviptadil resin 13.2g, resin rate of body weight gain 83.8%.
Embodiment 19: the preparation of aviptadil peptide crude product
Aviptadil resin 14.9g with embodiment 15 obtains joins in the 200ml flask, configuration 150ml lysate TFA:PhSMe:TIS:EDT:PhOH=85:3:3:4:5(V:V), lysate is joined in flask, room temperature reaction 2.5 hours, reaction finishes, filter resin, collect filtrate.With a small amount of TFA washing resin, merging filtrate, filtrate is joined in the 1500ml anhydrous diethyl ether precipitate, centrifugal, anhydrous diethyl ether washing, and vacuum-drying obtain aviptadil crude product 6.80g, wherein D-His 1-aviptadil 1.7%, D-Ser 2-aviptadil 3.3%, HPLC purity are 80.6%.Crude product yield 102.2%.MALDI-TOF：(M+H) +=3326.1。
Embodiment 20: the preparation of aviptadil peptide crude product
Aviptadil resin 14.9g with embodiment 15 obtains joins in the 200ml flask, configuration 150ml lysate TFA:PhSMe:TIS:EDT:PhOH=90:2:4:2:2(V:V), lysate is joined in flask, room temperature reaction 2.5 hours, reaction finishes, filter resin, collect filtrate.With a small amount of TFA washing resin, merging filtrate, filtrate is joined in the 1300ml anhydrous diethyl ether precipitate, centrifugal, anhydrous diethyl ether washing, and vacuum-drying obtain aviptadil crude product 6.86g, wherein D-His 1-aviptadil 1.5%, D-Ser 2-aviptadil 2.8%, HPLC purity are 83.6%.Crude product yield 103.2%, the HPLC collection of illustrative plates is seen Fig. 1.MALDI-TOF：(M+H) +=3326.3。
Embodiment 21(Comparative Examples): existing method prepares the aviptadil crude product
According to aviptadil peptide order one by one the synthetic aviptadil 2mmol of coupling method obtain aviptadil resin 11.5g, join in the 200ml flask, configuration 110ml lysate TFA:PhSMe:TIS:EDT:PhOH=90:1:4:2:3(V:V), lysate is joined in flask, room temperature reaction 2.5 hours, reaction finishes, and filters resin, collects filtrate.With a small amount of TFA washing resin, merging filtrate, filtrate is joined in the 1100ml anhydrous diethyl ether precipitate, centrifugal, anhydrous diethyl ether washing, and vacuum-drying obtain aviptadil crude product 6.16g, wherein D-His 1-aviptadil 6.0%, D-Ser 2-aviptadil 6.8%, HPLC purity are 75.4%.Thick peptide yield 92.7%, the HPLC collection of illustrative plates is seen Fig. 2.MALDI-TOF：(M+H) +=3326.0。
Embodiment 22: the mass-producing preparation of aviptadil peptide crude product
Aviptadil resin 800g with embodiment 15 obtains joins in the 10L reactor, configuration 8L lysate TFA:PhSMe:TIS:EDT:PhOH=90:2:4:2:2(V:V), lysate is joined in flask, room temperature reaction 2.5 hours, reaction finishes, filter resin, collect filtrate.With a small amount of TFA washing resin, merging filtrate, filtrate is joined in the 80L anhydrous diethyl ether precipitate, centrifugal, anhydrous diethyl ether washing, and vacuum-drying obtain aviptadil crude product 386.7g, thick peptide yield 108.3%.MALDI-TOF：(M+H) +=3326.6。
Embodiment 23: the purifying of aviptadil peptide crude product and turn acetate
After taking the mixed solvent 150mL ultrasonic dissolution of 14.6g aviptadil crude product with 10% acetonitrile water, adopt the Waters2545RP-HPLC system, wavelength 214nm, chromatographic column is the anti-phase C8 post of 50 * 250mm, conventional 0.1%TFA/ acetonitrile moving phase purifying, collection purpose peak cut obtains purity greater than 98.5% sterling.
Aviptadil sterling solution is adopted the RP-HPLC system, and chromatographic column is the anti-phase C18 post of 50 * 250mm, and 0.1% acetum/acetonitrile moving phase turns salt, collection purpose peak cut, rotary evaporation is concentrated, and freeze-drying obtains aviptadil acetate 5g, HPLC purity 99.4%, total recovery 33.86%.
The above is only the preferred embodiment of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Figure IDA00002967050200011
Figure IDA00002967050200021

Claims (12)

1. the method for a synthetic aviptadil, is characterized in that, comprises the following steps:
Step 1, solid phase synthesis have protecting group and hold coupling that the polypeptide fragment resin I of resin is arranged at C in coupling on aminoacid sequence Lys side chain shown in SEQ ID NO:1, on the Tyr side chain, on the Asn side chain, on the Ser side chain;
Solid phase synthesis coupling on the end of aminoacid sequence N shown in SEQ ID NO:2, Tyr side chain, on the Thr side chain, on the Arg side chain, on the Lys side chain, on the Gln side chain has protecting group and the polypeptide fragment resin II of resin is arranged in the coupling of C end;
Solid phase synthesis coupling on the end of aminoacid sequence N shown in SEQ ID NO:3, Asp side chain, on the Thr side chain, on the Asn side chain has protecting group and the polypeptide fragment resin III of resin is arranged in the coupling of C end, then the N end protecting group and Boc-His (3-Bum)-Ser (Psi (Me, Me) the pro)-OH coupling that remove the polypeptide fragment resin III obtain polypeptide fragment resin IV;
Step 2, add lysate to remove the resin of polypeptide fragment resin II and polypeptide fragment resin IV, correspondence obtains polypeptide fragment II and polypeptide fragment IV, then the C section of polypeptide fragment II and the N of polypeptide fragment resin I are held coupling, follow the N section coupling with C section with the polypeptide fragment II that removes N end protecting group of polypeptide fragment IV, obtain the aviptadil resin;
Step 3, add lysate to remove resin and all protecting groups in the aviptadil resin, obtain the aviptadil crude product, obtain the aviptadil sterling after the RP-HPLC purifying.
2. method according to claim 1, is characterized in that, the described solid-phase synthetic peptide fragment of step 1 resin I is:
Steps A 1, Fmoc-Asn(Trt)-OH carries out linked reaction with resin and obtains Fmoc-Asn(Trt under HOBt/DIPCDI double reagent coupling system or organic bases DIPEA effect)-resin;
Steps A 2, remove the Fmoc protecting group and obtain H-Asn(Trt)-resin, Fmoc-Leu-OH under the effect of HOBt/DIPCDI double reagent coupling system with H-Asn(Trt)-resin carries out linked reaction and obtains Fmoc-Leu-Asn(Trt)-resin;
steps A 3, hold the order of N end according to aminoacid sequence C shown in SEQ ID NO:1, successively one by one with Fmoc-Ile-OH, Fmoc-Ser (Trt)-OH, Fmoc-Asn (Trt)-OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Val-OH carries out amino acid according to steps A 2 coupling modes and extends coupling, remove at last N end Fmoc protecting group and obtain H-Val-Lys (Boc)-Lys (Boc)-Tyr (tBu)-Leu-Asn (Trt)-Ser (Trt)-Ile-Leu-Asn (Trt)-resin, it is polypeptide fragment resin I,
Wherein, described Fmoc is amino acid N end protecting group, and described tBu, Trt, Boc are the amino acid side chain protecting group.
3. method according to claim 1, is characterized in that, the described solid-phase synthetic peptide fragment of step 1 resin II is:
Step B1, Fmoc-Ala-OH carry out linked reaction with resin and obtain the Fmoc-Ala-resin under the DIPEA effect;
Step B2, remove the Fmoc protecting group and obtain the H-Ala-resin, Fmoc-Met-OH carries out linked reaction with the H-Ala-resin and obtains the Fmoc-Met-Ala-resin under the effect of HOBt/DIPCDI double reagent coupling system;
step B3, hold the order of N end according to aminoacid sequence C shown in SEQ ID NO:2, successively one by one with Fmoc-Gln (Trt)-OH, Fmoc-Lys (Boc)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Leu-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Thr (tBu)-OH, Fmoc-Tyr (tBu)-OH carries out amino acid according to step B2 coupling mode and extends coupling, obtain Fmoc-Tyr (tBu)-Thr (tBu)-Arg (Pbf)-Leu-Arg (Pbf)-Lys (Boc)-Gln (Trt)-Met-Ala-resin, it is polypeptide fragment resin II,
Wherein, described Fmoc is amino acid N end protecting group, and described tBu, Trt, Boc, Pbf are the amino acid side chain protecting group.
4. method according to claim 1, is characterized in that, the described solid-phase synthetic peptide fragment of step 1 resin III is:
Step C1, Fmoc-Asn (Trt)-OH carries out linked reaction with resin and obtains Fmoc-Asn (Trt)-resin under organic bases DIPEA effect;
Step C2, remove the Fmoc protecting group and obtain H-Asn (Trt)-resin, Fmoc-Asp (OtBu)-OH carries out linked reaction with H-Asn (Trt)-resin and obtains Fmoc-Asp (OtBu)-Asn (Trt)-resin under the effect of HOBt/DIPCDI double reagent coupling system;
Step C3, the order of holding the N end according to aminoacid sequence C shown in SEQ ID NO:3, one by one Fmoc-Thr (tBu)-OH, Fmoc-Phe-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Asp (OtBu)-OH are carried out amino acid according to step C2 coupling mode successively and extend coupling, obtain Fmoc-Asp (OtBu)-Ala-Val-Phe-Thr (tBu)-Asp (OtBu)-Asn (Trt)-resin, i.e. polypeptide fragment resin III;
step C4, DBLK removes the N end Fmoc protecting group of polypeptide fragment resin III, with Boc-His (3-Bum)-Ser (Psi (Me, Me) pro)-OH is at PyBOP/HOBt/DIPEA three reagent coupling systems, HOAt/DIPCDI double reagent coupling system, HOBt/DIPCDI double reagent coupling system, HATU/HOAt/DIPEA three reagent coupling systems, TBTU/HOBt/DIPEA three reagent coupling systems, or under HBTU/HOBt/DIPEA three reagent coupling system effects, coupling obtains Boc-His (3-Bum)-Ser (Psi (Me, Me) pro)-Asp (OtBu)-Ala-Val-Phe-Thr (tBu)-Asp (OtB u)-Asn (Trt)-resin, it is polypeptide fragment resin IV,
Wherein, described Fmoc, Boc are amino acid N end protecting group, and described tBu, OtBu, Trt are the amino acid side chain protecting group.
5. according to claim 1-4 described methods of any one, is characterized in that, described resin adopts substitution degree to be CTC resin, Rink Amide resin, Rink Amide-AM resin or the Rink Amide-MBHA resin of 0.2-1.0mmol/g.
6. according to claim 1-4 described methods of any one, is characterized in that, described coupling all one or both in DCM, NMP, DMF, the DMSO as solvent.
7. according to claim 2-4 described methods of any one, is characterized in that, the mol ratio of described each protected amino acid and HOBt, DIPCDI, DIPEA is 1:1.2:1.2:2.
8. method according to claim 4, it is characterized in that, the mol ratio of described Boc-His (3-Bum)-Ser (Psi (Me, Me) pro)-OH and HOBt, HOAt, HBTU, HATU, TBTU, PyBOP, DIPCDI, DIPEA is 1:1.2:1.2:1:1:1:1:1.2:2.
9. method according to claim 1, is characterized in that, the described polypeptide fragment II of step 3, polypeptide fragment IV and the mol ratio that removes the polypeptide fragment resin I of N segment protect base are 2-4:2-4:1.
10. method according to claim 1, is characterized in that, the described lysate of step 3 is that volume ratio TFE:DCM is the mixed pyrolysis liquid of 1:4.
11. method, is characterized in that according to claim 1, the described lysate of step 4 is that volume ratio TFA:PhSMe:TIS:EDT:PhOH is the mixed pyrolysis liquid cracking of 80-90:0-4:1-4:2-4:1-5.
12. method according to claim 1, it is characterized in that, PyBOP/HOBt/DIPEA three reagent coupling systems, HOAt/DIPCDI double reagent coupling system, HOBt/DIPCDI double reagent coupling system, HATU/HOAt/DIPEA three reagent coupling systems, TBTU/HOBt/DIPEA three reagent coupling systems or HBTU/HOBt/DIPEA three reagent coupling system couplings are adopted in the described coupling of step 2.
CN201310100852.3A 2013-03-26 2013-03-26 Method for synthetizing aviptadil Expired - Fee Related CN103159845B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201310100852.3A CN103159845B (en) 2013-03-26 2013-03-26 Method for synthetizing aviptadil

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201310100852.3A CN103159845B (en) 2013-03-26 2013-03-26 Method for synthetizing aviptadil

Publications (2)

Publication Number Publication Date
CN103159845A true CN103159845A (en) 2013-06-19
CN103159845B CN103159845B (en) 2015-03-18

Family

ID=48583409

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201310100852.3A Expired - Fee Related CN103159845B (en) 2013-03-26 2013-03-26 Method for synthetizing aviptadil

Country Status (1)

Country Link
CN (1) CN103159845B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103467566A (en) * 2013-09-13 2013-12-25 苏州维泰生物技术有限公司 Method for synthesizing novel pseudo dipeptide at kilogram level
CN104447963A (en) * 2014-11-14 2015-03-25 杭州阿德莱诺泰制药技术有限公司 Method for preparing aviptadil

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8178489B2 (en) * 2005-03-07 2012-05-15 Mondobiotech Ag Formulation for aviptadil
CN101472939B (en) * 2006-06-28 2012-10-10 弗·哈夫曼-拉罗切有限公司 Pseudo proline dipeptides

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8178489B2 (en) * 2005-03-07 2012-05-15 Mondobiotech Ag Formulation for aviptadil
CN101472939B (en) * 2006-06-28 2012-10-10 弗·哈夫曼-拉罗切有限公司 Pseudo proline dipeptides

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PETER C GROSS: "analytics of the therapeutic peptide aviptadil by sheathless CE-MS and comparison with namoRP-HPLC-MS", 《JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS》, 25 January 2014 (2014-01-25), pages 477 - 482, XP028783056, DOI: doi:10.1016/j.jpba.2013.09.024 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103467566A (en) * 2013-09-13 2013-12-25 苏州维泰生物技术有限公司 Method for synthesizing novel pseudo dipeptide at kilogram level
CN104447963A (en) * 2014-11-14 2015-03-25 杭州阿德莱诺泰制药技术有限公司 Method for preparing aviptadil

Also Published As

Publication number Publication date
CN103159845B (en) 2015-03-18

Similar Documents

Publication Publication Date Title
US11518794B2 (en) Synthesis method for liraglutide with low racemate impurity
CN103497245B (en) Method for synthesizing thymalfasin
CN102875665B (en) Method for synthesizing liraglutide
CN104004083B (en) A kind of method synthesizing Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]
CN106146648A (en) A kind of synthetic method of parathyroid hormone analogs
CN103224558B (en) A kind of preparation method of Exenatide
CN101747426B (en) Method for synthesizing pramlintide
CN104910269B (en) A method of synthesis Teriparatide
CN113880936B (en) Solid-phase synthesis method of abamectin
CN110590911A (en) Synthetic method and application of polypeptide containing tyrosine sulfating modification
CN104788546A (en) Preparation method of linear peptides containing 24 amino acid residues
CN104844706B (en) A method of synthesis lixisenatide
CN111748019A (en) Synthetic method of polypeptide derivative compound
CN106554391B (en) Method for synthesizing marine biological peptide Xen2174
CN102924575A (en) Preparation method of bivalirudin
WO2013078889A1 (en) The method for preparing exenatide by natural coupling
CN104072603A (en) Teduglutide synthesis method
CN103214568B (en) Solid phase method of secretin
CN112679602A (en) Solid-phase synthesis method of Somaloutide
CN109306366B (en) Method for synthesizing PT141
CN113754753A (en) Synthetic method of somaglutide
CN103159845B (en) Method for synthetizing aviptadil
CN104177491A (en) Preparation method for tesamorelin
CN113402598A (en) Solid-phase synthesis method of Somalutide
CN110615836B (en) Solid-phase synthesis method of liraglutide

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20150318

CF01 Termination of patent right due to non-payment of annual fee