CN103641891B - Method for preparing Kahalalide F - Google Patents
Method for preparing Kahalalide F Download PDFInfo
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- CN103641891B CN103641891B CN201310196587.3A CN201310196587A CN103641891B CN 103641891 B CN103641891 B CN 103641891B CN 201310196587 A CN201310196587 A CN 201310196587A CN 103641891 B CN103641891 B CN 103641891B
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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Abstract
The invention relates to a preparation method for Kahalalide F. The method comprises steps: 2-CTC resin is employed as a carrier, polypeptide segments A and B are synthesized through a solid phase polypeptide synthesis method; the polypeptide segment B is subjected to a molecule esterification reaction and a segment Bi is synthesized; the the polypeptide segments A and Bi are subjected to liquid phase condensation; protecting groups are removed and Kahalalide F is obtained. The method has high yield and convenient post-treatment, and provides a new ideal for industrial scale production of Kahalalide F.
Description
Technical field
The present invention relates to a kind of preparation method of polypeptide, and in particular to a kind of method for preparing Kahalalide F.
Background technology
Kahalalide F are one kind from sarcoglossan Mollusca Elysia rufescens and its food
Plumage algae（Bryopsis pennata）In the bioactive cyclic depsipeptides isolated（cyclic depsipetide）, its structure is such as
Shown in lower：
The structure is complicated, and which includes six aminoacid as loop section, and the ring exterior chain of seven amino acid is with acyl group
End-blocking.Kahalalide F are a kind of extremely rare and effective, source Yu Haiyang antineoplastic agents, and which is to carcinoma of prostate and knot
Colon-cancer cell shows good therapeutic effect, also shows certain biological activity in terms of antiviral, funguses.
Existing many documents describe the biological and pharmacological activity of kahalalide F, for example, be recorded in Hamann,
M.T.；Scheuer, P.J.J.Am.Chem.Soc.1993. " kahalalide F：One kind is from sarcoglossan Molluscas
Detached biological activity depsipeptides in Elysia refescens and chlorella Bryvopsis sp. ", volume 115,5825-5826
Page；Hamann, M.T. etc., J.Org.Chem.1996. " kahalalide：From sea mollusk Elysia
The biologically active peptide of rufescens and its algae food Bryops sp. ", volume 61, the 6594-6660 page；García-
Rocha, M. etc., Cancer Lett., 1996. " antitumoral compounds kahalalide F act on Cytolysosome ", the 99th
Volume, the 43-50 page.
United States Patent (USP) US2004/0067895A1 discloses a kind of comprising kahalalide F, non-ionic surfactant
The kahalalide F preparations of the freeze drying mixt of agent, organic acid and filler.
With regard to the preparation method of kahalalide F, the method that the method for report is mainly coupled one by one using solid phase at present
First synthesize peptide chain, then realize that intramolecular esterification obtains target molecule using the method for liquid phase synthesis, for example, be recorded in
Journal of Medicinal Chemistry,2008,51,4921；In Chinese patent CN1422278A.
However, needing in the reaction chain for gradually extending repeatedly deprotection in the technical process that solid phase is coupled one by one
Group, is so also easy to produce the impurity of difficult removing, and then makes intermediate reaction thing be difficult to obtain good purification, and causes final pure
Change complex loaded down with trivial details, cause yield low；Also, the cyclisation step that threonine side chains hydroxyl is coupled in route is easily caused finally
Product racemization, product configuration satisfactorily can not be controlled.
Have under the prospect of good medicine prospect in kahalalide F, need to develop a kind of low cost, high income, Yi Chun
The synthetic route of change is meeting ever-increasing scientific research and commercial production needs.
The content of the invention
For the deficiencies in the prior art, it is an object of the invention to provide a kind of new method for preparing kahalalide F, tool
Body provides a kind of method that solid phase fragment synthesis for preparing kahalalide F and liquid phase synthesis are combined, and which comprises the steps：
（1）With 2-CTC resins as carrier, polypeptide fragment A and B, wherein piece is respectively obtained using solid-phase peptide synthesis
Section A be：
Fragment B is：
Wherein, n1、n2、m1And m2It is 0 to 1 integer and the number of represented amino acid fragment；And while meet n1+m1
=1、n2+m2=1 and n1≥n2；
（2）In liquid phase environment, polypeptide fragment B is carried out into intramolecular esterification reaction and obtains following polypeptide fragment Bi:
（3）Polypeptide fragment A and Bi is made to carry out intramolecular coupling, deprotection base in liquid phase environment, obtain
kahalalide F。
The present invention prepares the method for kahalalide F and has following advantage compared to existing technology：
Synthetic route has transmutability；Reaction condition is gentle, simple to operate, post processing is easy, yield is high；And gained
Product configuration is single.This provides a kind of brand-new thinking for the large-scale production of kahalalide F.
Description of the drawings
Fig. 1 is the exemplified synthesis schemes that the present invention prepares kahalalide F.
Fig. 2 is chromatogram of the thick peptides of kahalalide F through RP-HPLC purification, in the spectrogram, retention time t=
It is the signal peak of kahalalide F at 17.012 minutes, peak area is 99.48%.
Specific embodiment
Term is explained
Term " solid-phase peptide synthesis " herein in the whole text refers to Fmoc solid phase synthesis sides known to Peptides Synthesis
Method, for example, be recorded in Fmoc Solid Phase Peptide Synthesis:A Practical Approach,
W.C.Chan, Peter D.White writes, March2, and 2000（ISBN-10:0199637245）, Britain Oxford
University Press。
Term " peptide resin " herein in the whole text refers to that the C-terminal of polypeptide is connected with resin, the amino of N-terminal is connected with Fmoc protection groups
Polypeptide resin.
Term " polypeptide fragment " herein in the whole text refers to that " peptide resin " mentioned above removing C-terminal resin exposes free carboxy
And the amino of N-terminal removes the polypeptide of Fmoc protection groups.
Used in specification and claims, the implication of english abbreviation is listed in the following table：
The invention provides a kind of method for preparing kahalalide F, which comprises the steps：
（1）With 2-CTC resins as carrier, polypeptide fragment A and B, wherein piece is respectively obtained using solid-phase peptide synthesis
Section A be：
Fragment B is：
Wherein, n1、n2、m1And m2It is 0 to 1 integer and the number of represented amino acid fragment；And while meet n1+m1
=1、n2+m2=1 and n1≥n2；
（2）In liquid phase environment, polypeptide fragment B is carried out into intramolecular esterification reaction and obtains following polypeptide fragment Bi：
（3）Polypeptide fragment A and Bi is made to carry out intramolecular coupling, deprotection base in liquid phase environment, obtain
kahalalide F。
Step of the present invention（1）Comprise the following steps
1）In the basic conditions, with 2-CTC resins as the first aminoacid of carrier conjugation：For fragment A is Fmoc-D-
AIIo-Ile-OH, Fmoc-L-Orn (Boc)-OH or Fmoc-D-Pro-OH and for fragment B be Fmoc-L-Val-OH, wherein
The alkali is DIPEA or TMP, preferably DIPEA.The substitution degree of 2-CTC resins is 0.2-1.0mmol/g, preferably 0.3-
0.8mmol/g。
2）Upper corresponding aminoacid is coupled successively using solid-phase peptide synthesis obtains peptide resin RAAnd RB, the solid phase
Polypeptide synthesis method includes：i）Removing Fmoc, then uses solvent washing resin, until detected with detection method removing completely
Till Fmoc；ii）By proper amount of after coupling amino acid and coupling agent dissolve in a solvent and activate, solid is added to together
In reaction column, till reaction terminating is detected with detection method；iii）Repeat i）And ii）.
The reagent for wherein removing Fmoc can be known in the art any reagent of the achievable purpose, preferably 20% piperazine
Pyridine/DMF solution（DBLK）, i.e. piperidines：DMF（Volume ratio）For 1:4 mixed solution.
Wherein need to use activator in Solid phase peptide synthssis, the activator used by the present invention is DIPCDI and compound I
Compositionss or DIPEA and compound I and compound II compositionss, wherein compound I is HOBt or HOAt, and compound II is
PyBOP, PyAOP, HATU, HBTU or TBTU.Wherein, in coupling agent the ratio of each composition with molar ratio computing as DIPCDI:I=1-
1.1:1, DIPEA:I:II=2:1:1.
Step 2）Reaction carry out in solid state reaction post.Solid state reaction post is not particularly limited, can be achievable this mesh
Any solid state reaction post.Additionally, the time that every kind of aminoacid carries out coupling reaction is usually 1-3 hours, preferably 1-2 is little
When；Pressure is preferably normal pressure, also can properly increase or the pressure that reduces under carry out；Temperature is preferably room temperature（I.e. 20 ± 5 DEG C）,
Also can carry out at a temperature of properly increasing or reducing.
Step 2）Reaction preferably resin is carried out before coupling it is swelling, it is described washing and swelling step this area can
Carried out using any reagent for realizing the purpose, preferably DMF.The detection method applied in the reaction be it is known in the art can
Any means of this purpose, such as chromatography or Chemical Calibration are realized, the reagent that can determine that reaction end is preferably used, preferably
1,2,3-indantrione monohydrate, when using 1,2,3-indantrione monohydrate, has free amide, i.e. amide nitrogen on unprotect base in illustrating polypeptide if resin develops the color.
In step 2）Used in aminoacid source for commercially available, wherein, Fmoc- (Z)-Dhb-OH is purchased from
Sigma-Aldrich, 5- methylhexanoic acid falls in love with extra large chemical conversion industry Development Co., Ltd purchased from ladder is uncommon, and remaining aminoacid is purchased from Shanghai
Gill is biochemical.
3）By peptide resin R in the presence of lysateAAnd RBFrom on resin, cracking obtains polypeptide fragment A and B.The cracking
Liquid is the TFE/DCM of 1% TFA/DCM or 20%（Percent by volume）.The combination of resulting polypeptide fragment A and B has following several
Kind：
n1=n2=0 and m1=m2In the case of=1
Fragment A1 is：
And fragment B1 is：
n1=1, n2=0 and m1=0, m2In the case of=1
Fragment A2 is：
And fragment B2 is：H-DaIle-DaThr-DaIle-DVal-LPhe-(Z)Dhb-LVal-OH
Or
n1=n2=1 and m1=m2In the case of=0
Fragment A3 is：
And fragment B3 is：PG-DaThr-DaIle-DVal-LPhe-(Z)Dhb-LVal-OH
PG=Bn, Boc or Fmoc
Wherein, for fragment B3, due to next needing to carry out intramolecular esterification, therefore the amino end of fragment B3
End is needed with blocking group（PG）, such as Bn, Boc or Fmoc protection group.
In step（1）The step of preferably also including polypeptide fragment A and B purification afterwards, the purification process can adopt ability
The method of purified polypeptide known to domain, such as chromatography, ion exchange or crystallization process；It is preferred that recrystallization method is adopted, specifically,
Using the system of THF/ petroleum ether, DCM/ petroleum ether or DCM/ normal hexane by fragment A and fragment B recrystallization.For the present invention,
The method is easily operated, and product yield is high, and spatial configuration retentivity is preferable.
In the step of the present invention（2）In, polypeptide fragment B obtains fragment Bi through intramolecular esterification Jing after cracking
What above-mentioned intramolecular esterification reaction was carried out in the presence of coupling agent.The coupling agent is DIPCDI and compound I
Compositionss or DIPEA and compound I, wherein compound I be HOBt or HOAt.Further, in coupling agent each composition ratio
Example is with molar ratio computing as DIPCDI:I=1.1:1.0.
Above-mentioned intramolecular esterification reaction is carried out in, and the response time is 3-5 hours.Reaction end can be using prior art
The means known detecting, such as thin layer chromatography, HPLC methods etc., it is preferred to use HPLC methods.
Step of the present invention（3）Carry out in the presence of coupling agent.The coupling agent is DIPEA and compound I
With the compositionss of compound II, wherein compound I be HOBt or HOAt, compound II be PyBOP, PyAOP, HATU, HBTU or
TBTU.Further, in coupling agent the ratio of each composition with molar ratio computing as DIPEA:I:II=2.0:1.1:1.0.
For polypeptide fragment Bi-3, needed first to experience removing amino end before the coupling reaction with polypeptide fragment A3 is carried out
The reaction of end protection group：
For Boc protection groups, the reagent for adopting for 80% TFA/DCM（Percent by volume）Mixed solution；
For Bn protection groups, the reagent for adopting for 5% palladium carbon, carry out hydro-reduction reaction in hydrogen atmosphere；
For Fmoc protection groups, the reagent for adopting is for 1:7 piperidines/DCM mixed solutions（Volume ratio）.
Step（3）In, the reagent of deprotection base is the mixed solution of TFA or HCl and PhOMe or DCM, preferably TFA with
The mixed solution of DCM, further, TFA:DCM=95:5（Volume ratio）.
In step（3）Afterwards, purification step of the present invention preferably also including kahalalide F.The purification step can be adopted
Any polypeptide purification techniques known in the art are carried out, it is preferred to use reversed-phase high pressure liquid chromatography.Further, it is described anti-phase
High pressure lipuid chromatography (HPLC) includes：It is with anti-phase octadecylsilane as fixing phase, pure with 0.05%TFA/ water, acetonitrile mobile phase system
Change, collect purpose peak fraction, it is concentrated freeze-dried to obtain kahalalide F fine peptides.
Embodiment
For a further understanding of the present invention, carry out specifically with reference to specific embodiment and referring to the drawings 1 couple of present invention
It is bright, it should be appreciated that following embodiments are intended to explanation, do not limit the invention.
The 2-CTC resins for using in this manual are purchased from Tianjin Nankai and into company limited, Fmoc- (Z)-Dhb-OH purchases
Extra large chemical conversion industry Development Co., Ltd is fallen in love with from Sigma-Aldrich, 5- methylhexanoic acid purchased from ladder is uncommon, remaining aminoacid is purchased from upper
Extra large gill is biochemical；Coupling reagent（Activator）Purchased from Suzhou Mantidiss company limited, other solvents and reagent are common commercially available product.
Embodiment 1：The preparation of Fmoc-D-Pro-CTC resins
Weigh dry 2-CTC resin 221g（Substitution degree is 0.736mmol/g）It is added in solid state reaction post, uses first
DMF washing resins 2 times, then with the DMF swellable resins 1 hour of 2-3 times of resin bed volume, then washed with DMF successively 3 times,
DCM is washed 2 times, is continued to employ.
Under conditions of ice bath cooling, 109.8g Fmoc-D-Pro-OH, 42.1g DIPEA are dissolved in into the mixed of DMF and DCM
In bonding solvent, after aminoacid dissolving, activated Fmoc-D-Pro-OH is added to into reaction 2 hours in reaction column slowly, instead
After should terminating, add in reaction column and reacted 30 minutes by the confining liquid that 100mL methanol and 21.0g DIPEA are constituted.DMF is washed
After 3 times, that is, obtain Fmoc-D-Pro-CTC resins.
Embodiment 2：The preparation of Fmoc-L-Orn (Boc)-CTC resins
Weigh dry 2-CTC resin 288g（Substitution degree is 0.534mmol/g）It is added in solid state reaction post, uses first
DMF washing resins 2 times, then with the DMF swellable resins 1 hour of 2～3 times of resin bed volumes, then washed with DMF successively 3 times,
DCM is washed 2 times, is continued to employ.
Under conditions of ice bath cooling, 139.8g Fmoc-L-Orn (Boc)-OH, 39.8gDIPEA are dissolved in into DMF and DCM
Mixed solvent in, after aminoacid dissolving after, slowly activated Fmoc-L-Orn (Boc)-OH is added in reaction column instead
Answer 2 hours, after reaction terminates, add in reaction column and 30 points are reacted by the confining liquid that 95mL methanol and 20.0g DIPEA are constituted
Clock.After DMF washs 3 times, that is, obtain Fmoc-L-Orn (Boc)-CTC resins.
Embodiment 3：The preparation of Fmoc-D-Allo-Ile-CTC resins
Weigh dry 2-CTC resin 345g（Substitution degree is 0.385mmol/g）It is added in solid state reaction post, uses first
DMF washing resins 2 times, then with the DMF swellable resins 1 hour of 2～3 times of resin bed volumes, then washed with DMF successively 3 times,
DCM is washed 2 times, is continued to employ.
Under conditions of ice bath cooling, 93.9g Fmoc-D-Allo-Ile-OH, 34.4gDIPEA are dissolved in into DMF and DCM
Mixed solvent in, after aminoacid dissolving after, slowly activated Fmoc-D-Allo-Ile-OH is added in reaction column instead
Answer 2 hours, after reaction terminates, add in reaction column and 30 points are reacted by the confining liquid that 90mL methanol and 17.0g DIPEA are constituted
Clock.After DMF washs 3 times, that is, obtain Fmoc-D-Allo-Ile-CTC resins.
Embodiment 4：The preparation of Fmoc-L-Val-CTC resins
Weigh dry 2-CTC resin 316g（Substitution degree is 0.385mmol/g）It is added in solid state reaction post, uses first
DMF washing resins 2 times, then with the DMF swellable resins 1 hour of 2～3 times of resin bed volumes, then washed with DMF successively 3 times,
DCM is washed 2 times, is continued to employ.
Under conditions of ice bath cooling, 82.6g Fmoc-L-Val-OH, 31.5g DIPEA are dissolved in into the mixed of DMF and DCM
In bonding solvent, after aminoacid dissolving, activated Fmoc-L-Val-OH is added to into reaction 2 hours in reaction column slowly, instead
After should terminating, add in reaction column and reacted 30 minutes by the confining liquid that 86mL methanol and 15.8g DIPEA are constituted.DMF washings 3
After, that is, obtain Fmoc-L-Val-CTC resins.
Embodiment 5：Peptide resin RA1Synthesis
Fmoc-D-Pro-CTC resins in Example 1 are added in reactor, swelling 0.5 hour with DMF, then are used
20%DBLK removes Fmoc protections twice, detects the color of resin with ninhydrin method, after being washed with DMF, is coupled Fmoc-D-Val-
OH.By 110.5gFmoc-Val-OH, 44g HOBt, 41g DIPCDI（Or 41.8g HOAt, 111g HATU and 79.5g
DIPEA）It is dissolved in DMF, in ice-water bath, pre-activate was added in solid phase reactor after 5 minutes, room temperature reaction 1-2 hours.With
Ninhydrin method detection judges reaction end.The step of Fmoc of removing more than repeating, continue to be coupled upper Fmoc-L-Val-OH, Fmoc-
L-Thr (tBu)-OH and Fmoc-D-Val-OH, then swelling 1.0 hours with DMF, then remove Fmoc guarantors twice with 20%DBLK
Shield, detects the color of resin with ninhydrin method, after being washed with DMF, starts to connect 5-MeHex-OH.By 43.3g5-MeHex-OH,
44g HOBt, 41g DIPCDI is dissolved in DMF, in ice-water bath after pre-activate 5min, in adding solid phase reactor, room temperature reaction
1～2 hour.Reaction end is judged with ninhydrin method detection.Fmoc protections, DMF is removed twice with 20%DBLK after completion of the reaction
Washing, methanol are shunk 3 times, and removal of solvent under reduced pressure obtains peptide resin RA1。
Peptide resin R can be synthesized using similar methodA2And RA3。
Embodiment 6：The synthesis of peptide resin B
Fmoc-L-Val-CTC resins in Example 4 are added in reactor, swelling 0.5 hour with DMF, then are used
20%DBLK removes Fmoc protections twice, detects the color of resin with ninhydrin method, after being washed with DMF, starts to connect Fmoc-Z-
Dhb-OH.78.7g Fmoc-Z-Dhb-OH, 32.9g HOBt, 29.6g DIPCDI are dissolved in DMF, the pre- work in ice-water bath
After changing 5 minutes, in adding solid phase reactor, room temperature reaction 1-2 hours.Reaction end is judged with ninhydrin method detection.Repeat with
The step of upper removing Fmoc, continue to be coupled upper Fmoc-L-Phe-OH, Fmoc-D-Val-OH, Fmoc-D-Allo-Ile-OH,
The coupling of Fmoc-D-Allo-Thr-OH, Fmoc-D-Allo-Ile-OH and Fmoc-L-Orn (Boc)-OH aminoacid, obtains peptide
Resin RB1。
Peptide resin R can be synthesized using similar methodB2。
Embodiment 7：Peptide resin RB3The synthesis of peptide resin of the middle amino terminal with different protection groups
The coupling of Boc-D-Allo-Thr-OH
Fmoc-D-Allo-Ile-DVal-LPhe- (Z) Dhb-LVal-CTC resins will have been linked to put in embodiment 6
In reaction column, swelling 1.0 hours with DMF, then Fmoc protections are removed twice with 20%DBLK, detect resin with ninhydrin method
Color, after being washed with DMF, starts to connect Boc-D-Allo-Thr-OH.By 53.4gBoc-D-Allo-Thr-OH, 33.8g
HOAt, 30.9g DIPCDI is dissolved in DMF, the pre-activate after 5 minutes in ice-water bath, in adding solid phase reactor, room temperature reaction 1
～2 hours.Reaction end is judged with ninhydrin method detection.DMF washings after completion of the reaction, methanol are shunk 3 times, removal of solvent under reduced pressure
Obtain the D type threonine peptide resin R containing Boc protectionsB3。
The coupling of Bn-D-Allo-Thr-OH
Fmoc-D-Allo-Ile-DVal-LPhe- (Z) Dhb-LVal-CTC resins will have been linked to put in embodiment 6
In reaction column, swelling 1.0 hours with DMF, then Fmoc protections are removed twice with 20%DBLK, detect resin with ninhydrin method
Color, after being washed with DMF, starts to connect Bn-D-Allo-Thr-OH.By 51.2gBn-D-Allo-Thr-OH, 32.9g HOBt,
74.2g TBTU, 62.8g DIPEA is dissolved in DMF, in ice-water bath after pre-activate minute, in adding solid phase reactor, room temperature
Reaction 1～2 hour.Reaction end is judged with ninhydrin method detection.Washed with DMF after completion of the reaction, methanol shrinks 3 times, decompression
Solvent is removed, the D type threonine peptide resin R containing benzyl protection can be obtainedB3。
The coupling of Fmoc-D-Allo-Thr-OH
Fmoc-D-Allo-Ile-DVal-LPhe- (Z) Dhb-LVal-CTC resins will have been linked to put in embodiment 6
In reaction column, swelling 1.0 hours with DMF, then Fmoc protections are removed twice with 20%DBLK, detect resin with ninhydrin method
Color, after being washed with DMF, starts to connect Fmoc-D-Allo-Thr-OH.By 83.1gFmoc-D-Allo-Thr-OH, 33.8g
HOAt, 120.3g PyBOP, 59g TMP is dissolved in DMF, in ice-water bath after pre-activate 5min, in adding solid phase reactor, room
Temperature reaction 1～2 hour.Reaction end is judged with ninhydrin method detection.Washed with DMF after completion of the reaction, methanol shrinks 3 times, subtracts
Pressure removes solvent, can obtain the D type threonine peptide resin B3 containing Fmoc protections.
Embodiment 8：Peptide resin RAAnd RBCracking
By dry peptide resin RA3（339g）It is placed in cracking reaction kettle, adds 20% trifluoroethanol/DCM mixed solutions
3.4L（Cracking reaction is carried out according to 1g resin 10mL lysates proportionings）, room temperature reaction 2h is filtered to remove resin after completion of the reaction,
Liquor capacity is concentrated into 400mL by concentrating under reduced pressure, and polypeptide fragment A3 about 123g, MALDI- is obtained with the ether sedimentation of 4L then
TOF：(M+H)+=1009.7。
By dry peptide resin RB3（Amino terminal band Boc protection groups）（451g）It is placed in cracking reaction kettle, adds 1%
TFA/DCM mixed solution 4.5L（Cracking reaction is carried out according to 1g resin 10mL lysates proportionings）, room temperature reaction 2h, reaction are finished
After be filtered to remove resin, liquor capacity is concentrated into 500mL by concentrating under reduced pressure, and polypeptide fragment is obtained with the sedimentation of the ether of 5L then
B3 about 136g, MALDI-TOF：(M+H)+=883.6。
Embodiment 9：The recrystallization of polypeptide fragment A3 and B3
The thick peptide 60g for taking the polypeptide fragment A3 that cracking is obtained is placed in the round-bottomed flask of 1L, adds 300mL tetrahydrofurans,
Reflux state is heated to slowly, after thick peptide all dissolves（If failing dissolving, continue to add a small amount of tetrahydrofuran until dissolving）,
Slowly Deca petroleum ether, when in solution having white solid to separate out, stops adding petroleum ether, slowly quiet in the environment of placing 5 DEG C
Put overnight, decompression sucking filtration obtains 51.4g purity more than 95%, single miscellaneous polypeptide fragment A3 below 0.5%.
The thick peptide 70g for taking the polypeptide fragment B3 that cracking is obtained is placed in the round-bottomed flask of 1L, adds 380mL DCM, slowly
Reflux state is heated to, after thick peptide all dissolves（If failing dissolving, continue to add a small amount of DCM until dissolving）, slowly Deca
Petroleum ether, when in solution having white solid to separate out, stops adding petroleum ether, slowly stood in the environment of being positioned over 0-5 DEG C
Night, decompression sucking filtration are obtained 58.1g purity more than 95%, single miscellaneous polypeptide fragment B3 below 0.5%.
Embodiment 10：The synthesis of the polypeptide fragment Bi-3 with protection group
The polypeptide fragment B3 of Jing recrystallization purifyings in Example 9（27g）, dissolved with the DCM of 1000mL, ice bath cooling,
The temperature of control reaction system adds 3.8g HOBt between 0-5 DEG C, after HOBt all dissolves, slowly Deca 4mL
DIPCDI, keeps reaction temperature to react about 12 hours between 0-5 DEG C, HPLC tracking reactions, and after reaction terminates, adding water, it is anti-to be quenched
Should, DCM extractions, organic faciess use 1mol/L hydrochloric acid, the sodium bicarbonate solution of saturation, saturated nacl aqueous solution to wash successively, anhydrous sulfur
Sour sodium is dried, and concentrating under reduced pressure is obtained polypeptide fragment Bi-3s of the 25.4g with protection group, MALDI-TOF：(M+H)+=865.5,
Need not be further purified and be directly used in reaction below.
Embodiment 11：The synthesis of polypeptide fragment Bi-3
The polypeptide fragment Bi-3 for taking 21g Boc protections is directly dissolved in the TFA/DCM mixed solvents of 300mL80%, and room temperature is stirred
Reaction 2 hours is mixed, HPLC detections are reacted, and after the completion of question response, reduce pressure away solvent, then is dissolved with the DCM of 50mL or so,
The absolute ether precipitation of 1L, you can obtain 18.6g polypeptide fragment Bi-3.
The polypeptide fragment Bi-3 of 25.4g Fmoc protections is taken, is dissolved in the DCM of 350mL, added 50mL piperidines, be stirred at room temperature
Reaction 2 hours, HPLC detection reactions, after the completion of question response, reduce pressure away solvent, then is dissolved with the DCM of 50mL or so, 1L
Absolute ether precipitation, obtain 20.9g polypeptide fragment Bi-3.
The polypeptide fragment Bi-3 of 20g Bn protections is taken, is dissolved in the DCM of 300mL, add the palladium carbon powder that content is 5%
0.5g, the room temperature reaction 3h in atmosphere of hydrogen, HPLC detection reaction, after the completion of question response, is filtered to remove palladium charcoal, reduces pressure away molten
Agent, is dissolved with the DCM of 50mL or so, the absolute ether precipitation of 1L, obtains 17.4g polypeptide fragment Bi-3.
Embodiment 12：The coupling of polypeptide fragment A3 and Bi-3
Polypeptide fragment A3 and Bi-3 are used for into the reaction.33.6g polypeptide fragment A3 and 20.9g polypeptide fragment Bi-3 are taken respectively
It is placed in round-bottomed flask, adds the DCM of 300mL, after fragment all dissolves, with ice bath by the temperature control of reaction system in 0-
Between 5 DEG C, then it is slowly added into 4.4g HOAt, 11.2g HATU and 7.7g DIPEA in flask successively, is kept for 0-5 DEG C
30min, is then back to room temperature reaction 4 hours or so, HPLC tracking reactions, after question response terminates, adds water and reaction is quenched, DCM extractions
Take, organic faciess use 1mol/L hydrochloric acid, the sodium bicarbonate solution of saturation, saturated nacl aqueous solution to wash successively, anhydrous sodium sulfate drying,
It is concentrated under reduced pressure to give crude product 50.8g, MALDI-TOF：(M+H)+=1634.2。
Embodiment 13：The preparation of the thick peptides of Kahalide F
The crude product obtained in embodiment 12 is directly dissolved in the TFA/DCM solution of 500mL95%, room temperature reaction 2 is little
When, HPLC detection reactions, after the completion of question response, reduce pressure away solvent, then is dissolved with the DMF of 80mL or so, the steaming of 1.5L
Distilled water is precipitated, and sucking filtration, drying under reduced pressure obtain the thick peptide 43.5g of Kahalide F, HRMS m/z:found(M+H)+=1477.9328。
Embodiment 14：The preparation of Kahalide F fine peptides
The thick peptide 43.5g of Kahalide F obtained in Example 13 are dissolved in 300mL acetonitriles, using NOVASEP
RP-HPLC systems, wavelength 220nm, chromatographic column are anti-phase C18 posts, and conventional 0.05%TFA/ water, acetonitrile mobile phase system purification are removed
Salt, collects purpose peak fraction, and rotary evaporation concentration, lyophilizing obtain Kahalide F fine peptides 28.3g, HPLC purity 99.48%.
Although describing the present invention with reference to particular, those skilled in the art will recognize that
It is, in the case of without departing from spirit and scope of the present invention, the embodiment can be changed or be improved, the scope of the invention
Limited by appended claims.
Claims (13)
1. a kind of method for preparing kahalalide F, the method comprise the steps：
(1) with 2-CTC resins as carrier, polypeptide fragment A and B, wherein fragment A are respectively obtained using solid-phase peptide synthesis
For：
Fragment B is：
Wherein, n1、n2、m1And m2It is 0 to 1 integer and the number of represented amino acid fragment；And while meet n1+m1=1,
n2+m2=1 and n1≥n2；
Wherein, first in the basic conditions with 2-CTC resins as carrier conjugation on first aminoacid：For fragment A is Fmoc-D-
AIIo-Ile-OH, Fmoc-L-Orn (Boc)-OH or Fmoc-D-Pro-OH and for fragment B be Fmoc-L-Val-OH；Then
Upper corresponding aminoacid is coupled successively using solid-phase peptide synthesis obtains peptide resin RAAnd RB；The last presence in lysate
It is lower by peptide resin RAAnd RBFrom on resin, cracking obtains polypeptide fragment A and B, and polypeptide fragment A and B is carried out using recrystallization method
Purification, wherein for n1=n2=1 and m1=m2=0 situation, the amino terminal of polypeptide fragment B3 carry blocking group；
(2) in liquid phase environment, polypeptide fragment B is carried out into intramolecular esterification reaction and obtains following polypeptide fragment Bi:
(3) polypeptide fragment A and Bi is made to carry out intramolecular coupling, deprotection base in liquid phase environment, obtain kahalalide F.
2. the method for claim 1 wherein that the blocking group that the amino terminal of fragments of peptides B3 is carried is protected for Bn, Boc or Fmoc
Base.
3. the method for claim 1 wherein that the reaction of step (1) is carried out in the basic conditions, the substitution degree of 2-CTC resins is
0.2-1.0mmol/g；The alkali for using is DIPEA or TMP.
4. the method for claim 3, the wherein substitution degree of 2-CTC resins are 0.3-0.8mmol/g.
5. the method for claim 3, the alkali used in which are DIPEA.
6. the method for claim 1 wherein that step (2) is carried out in the presence of an activator, the activator is DIPCDI and change
The compositionss of the compositionss of compound I or DIPEA and compound I and compound II, wherein compound I be HOBt or HOAt, chemical combination
Thing II is PyBOP, PyAOP, HATU, HBTU or TBTU, and wherein, in coupling agent, the ratio of each composition with molar ratio computing is
DIPCDI:I=1-1.1:1, DIPEA:I:II=2:1:1.
7. the method for claim 1 wherein that step (3) is carried out in the presence of lysate, the lysate is with percent by volume
It is calculated as 1% TFA/DCM or 20% TFE/DCM.
8. the method for claim 1 wherein the recrystallization method using THF/ petroleum ether, DCM/ petroleum ether or DCM/ normal hexane
Mixed solution.
9. the method for claim 1 wherein that the intramolecular esterification of step (2) reacts what is carried out in the presence of coupling agent, it is described
Coupling agent for DIPCDI and compound I compositionss, wherein compound I be HOBt or HOAt, the ratio of each composition in coupling agent
With molar ratio computing as DIPCDI:I=1.1:1.0.
10. the method for claim 1 wherein that step (3) is carried out in the presence of coupling agent, the coupling agent is DIPEA
With compound I and the compositionss of compound II, wherein compound I be HOBt or HOAt, compound II be PyBOP, PyAOP,
HATU, HBTU or TBTU, in coupling agent, the ratio of each composition is with molar ratio computing as DIPEA:I:II=2.0:1.1:1.0.
11. the method for claim 1 wherein mixed solution that the reagent of deprotection base in step (3) is TFA and DCM, wherein
Mixed solution is with volume basis as TFA:DCM=95:5.
The method of 12. claim 1, also including the purification step of kahalalide F.
The method of 13. claim 12, wherein purification adopt reversed-phase high pressure liquid chromatography, including：With anti-phase octadecyl silicon
Alkane is fixing phase, with 0.05%TFA/ water, acetonitrile mobile phase system purification, collects purpose peak fraction, concentrated freeze-dried to obtain
Kahalalide F fine peptides.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1422278A (en) * | 2000-02-09 | 2003-06-04 | 法马马有限公司 | Kahalalide F and related compounds |
| WO2005103072A1 (en) * | 2004-04-22 | 2005-11-03 | Pharma Mar, S.A.U. | Convergent synthesis for kahalalide compounds |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1422278A (en) * | 2000-02-09 | 2003-06-04 | 法马马有限公司 | Kahalalide F and related compounds |
| WO2005103072A1 (en) * | 2004-04-22 | 2005-11-03 | Pharma Mar, S.A.U. | Convergent synthesis for kahalalide compounds |
Non-Patent Citations (1)
| Title |
|---|
| Convergent Approaches for the Synthesis of the Antitumoral Peptide, Kahalalide F. Study of Orthogonal Protecting Groups;Carolina Gracia et al;《The Journal of Organic Chemistry》;20060930;第71卷(第19期);图1，第7197右栏第1-5段、第7198左栏第1-5段、第7199页SCHEME4，第7200页左栏第3-6段，表1，第7199页strategy4，第7200左栏第2段，第7020页 * |
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