CN103351428A - Synthesis of degarelix by solid phase segment method - Google Patents
Synthesis of degarelix by solid phase segment method Download PDFInfo
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- CN103351428A CN103351428A CN2013103364467A CN201310336446A CN103351428A CN 103351428 A CN103351428 A CN 103351428A CN 2013103364467 A CN2013103364467 A CN 2013103364467A CN 201310336446 A CN201310336446 A CN 201310336446A CN 103351428 A CN103351428 A CN 103351428A
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- 0 CC(C(Cc1cc(cccc2)c2cc1)*=C)=O Chemical compound CC(C(Cc1cc(cccc2)c2cc1)*=C)=O 0.000 description 3
- RZXMPPFPUUCRFN-UHFFFAOYSA-N Cc(cc1)ccc1N Chemical compound Cc(cc1)ccc1N RZXMPPFPUUCRFN-UHFFFAOYSA-N 0.000 description 1
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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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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention discloses synthesis of degarelix by a solid phase segment method, wherein Fmoc-amino acid is connected with resin so that Fmoc-amino acid-resin can be obtained; a solid phase synthesis method is adopted, and the steps of orderly connecting amino acid from the end C to the end N and removing the Fmoc group are carried out, so that Fmoc protected polypeptide resin can be obtained; the method comprises the following steps of: (1) forming polypeptide resin as shown in formula III and polypeptide resin as shown in formula IV, respectively; (2) mixing the polypeptide resin as shown in formula III with 1-5% of trifluoroacetic acid (TFA), connecting L-4,5-dihydroorotate to the side chain amino of the polypeptide resin after the protecting group of the sixth amino acid residue at the end C is removed, and then removing the Fmoc group to obtain polypeptide resin as shown in formula V; removing the Fmoc group of the polypeptide resin as shown in formula IV and acetylating the polypeptide resin, thereby obtaining polypeptide resin as shown in formula VI, and then cutting to obtain segments as shown in formula VII; (3) coupling the polypeptide resin as shown in the formula V with the segments as shown in the formula VII, thereby obtaining the polypeptide resin as shown in formula II; (4) separating polypeptide on the polypeptide resin as shown in the formula II from the resin, thereby obtaining degarelix as shown in formula I.
Description
Technical field
The present invention relates to the synthetic field of Solid-phase Polypeptide, particularly the synthetic Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 of solid phase fragment method.
Background technology
Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is a kind of GnRH antagonist, the medicine of a kind of new treatment advanced prostate cancer of being developed by Ferring (brightness icepro) company, and commodity are called Firmagon.The Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 onset is rapid, suppress gonadotropin, testosterone and prostate specific antigen, its effect that reduces testosterone concentration can compare favourably with Leuprolide depot controlled release injection (Lupron Depot) at least, and reduces testosterone concentration and want statistically significantly fast.
Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is the decapeptide of synthetic, and wherein major part is the alpha-non-natural amino acid of D type, and its peptide order structure is
Ac-D-2Nal-D-Phe (4Cl)-D-3Pal-Ser-4Aph (Hor)-D-4Aph (Cbm)-Leu-Lys (ipr)-Pro-D-Ala-NH
2, chemical structural formula is as follows:
The side chain amino protecting group L-Hor(L-hydroorotic acid of the 6th amino-acid residue 4Aph (L-Hor) in the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 from the C end) contains the dihydrouracil structure in.Document patent report (Koedjikov, A.H.et.al., J.Chem.Soc.Perkin, Trans.2,1984,1077-1081 are arranged; Kaneti, J.et.al., Org.Biomol.Chem., 2004,1098-1103; WO2010121835): the dihydrouracil structure among the six-ring Hor is easily reset under alkaline condition and is formed the glycolylurea structure.After can causing Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 to be reset, above-mentioned reaction can produce glycolylurea similar thing impurity A c-D-2Nal-D-Phe (4Cl)-D-3Pal-Ser-4Aph (Z)-D-4Aph (Cbm)-Leu-Lys (ipr)-Pro-D-Ala-NH in building-up process
2, wherein Z represents glycolylurea-5-ethanoyl.Because character and the product Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 of the similar thing impurity of glycolylurea are similar, polarity is close, thereby has greatly increased the purifying difficulty of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude product, has also additionally increased its industrial production cost.
Brightness is insulted and adopted the earliest Boc solid phase synthesis strategy in patent US5925730, adopts trifluoroacetic acid to remove the Boc protecting group.Although effectively avoid the generation of the similar thing impurity of glycolylurea, Boc solid phase synthesis strategy need to adopt HF when the peptide resin cracking, and HF has larger harm to human and environment, is difficult to carry out large-scale production.
Patent WO2010121835 and WO2011066386 have all adopted Fmoc solid phase synthesis strategy, and wherein the 6th amino acid adopts Fmoc-4Aph (L-Hor)-OH.Take aminoresin as carrier, connect successively 10 amino acid among the patent WO2010121835, adopt 20%PIP/DMF solution to remove the Fmoc protecting group, can effectively control in the similar thing impurity of the glycolylurea 0.1-0.3% scope; Adopted the segment condense strategy of solid liquid phase combination among the patent WO2011066386; provide 9+1 fragment condensation and 3+6+1 fragment condensation two kinds of methods; but also need under alkaline condition, remove the Fmoc protecting group, be difficult to the generation that the similar thing of impurity glycolylurea is reset in effectively control.
The 6th amino acid adopts Fmoc-4Aph (X)-OH among the patent CN102329373, and wherein X represents Trt or Alloc.After adopting Fmoc solid phase synthesis strategy to connect successively amino acid and acetylize; remove again the protecting group X of amino-acid residue 4Aph (X); connect L-Hor (L-4 at its side chain amino; the 5-dihydroorotate), reduce Hor and under alkaline condition, reset the possibility that produces the similar thing impurity of glycolylurea.Yet adopt 5-10%TFA/DCM when removing the protecting group Trt of amino-acid residue 4Aph (Trt), can cause the 3rd amino-acid residue Lys (ipr, Boc) part of protecting group Boc comes off, thus exposed amino meeting and L-4, and the reaction of 5-dihydroorotate generates new impurity; Adopt Pd (Ph when in addition, removing the protecting group Alloc of amino-acid residue 4Aph (Alloc)
3P)
4/ phenylsilane/DCM, Pd (Ph
3P)
4Price comparison expensive, can increase the cost of large-scale production.
Therefore, this area is in the urgent need to providing a kind of technique of new solid phase synthesis Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2.
Summary of the invention
The present invention aims to provide a kind of technique of new solid phase synthesis Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2.
The invention provides a kind of solid phase synthesis preparation method thereof suc as formula the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 shown in the I; Fmoc-amino acid is connected with resin; obtain Fmoc-amino acid-resin; adopt the step of method through amino acid is held in order successively connection and the Fmoc group is removed to N from the C end of solid phase synthesis; obtain the polypeptide resin of Fmoc protection, described method comprises step:
(1) forms respectively suc as formula the polypeptide resin shown in III and the formula IV;
(2) will mix suc as formula the trifluoracetic acid (TFA) of the polypeptide resin shown in the III and 1-5%, after sloughing the protecting group X that C holds the 6th amino-acid residue, connect L-4 at its side chain amino, the 5-dihydroorotate removes the Fmoc group and obtains suc as formula the polypeptide resin shown in the V;
To remove Fmoc group, acetylize suc as formula the polypeptide resin shown in the IV obtains obtaining suc as formula the fragment shown in the VII suc as formula cutting behind the polypeptide resin shown in the VI;
(3) will carry out coupling by solid phase method suc as formula the polypeptide resin shown in the V with suc as formula the fragment shown in the VII, obtain suc as formula the polypeptide resin shown in the II; With
(4) polypeptide and the resin isolation on the polypeptide resin shown in the formula II obtains suc as formula the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 shown in the I;
Wherein X is selected from Mmt or Dmt; Preferred Mmt.
In another preference, be selected from Rink Amid-MBHA resin or Rink Amide-AM resin suc as formula the resin in the polypeptide resin shown in the III, substitution degree is 0.3-0.7mmol/g; Substitution degree suc as formula the resin CTC in the polypeptide resin shown in the IV is 0.8-1.2.
In another preference; the amino acid of the protection of formation during suc as formula the polypeptide resin shown in the III is respectively Fmoc-D-Ala-OH, Fmoc-Pro-OH, Fmoc-Lys (ipr, Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4Aph (Cbm)-OH, Fmoc-4Aph (X)-OH.
In another preference, the amino acid of the protection when forming suc as formula the polypeptide resin shown in the IV be respectively Fmoc-Ser (
tBu)-OH, Fmoc-D-3Pal-OH, Fmoc-D-Phe (4Cl)-OH, Fmoc-D-2Nal-OH.
In another preference, the coupling agent of use is selected from DIC/HOBt, DIC/Cl-HOBt, DIC/HOAt, TBTU/HOBt/DIEA, TBTU/Cl-HOBt/DIEA, PyBop/HOBt/DIEA or PyBop/Cl-HOBt/DIEA.
In another preference, the linked reaction solvent is DMF, NMP and DMF/DCM mixed solvent; Temperature of reaction is room temperature, and the reaction times is for stirring 2-4h.
In another preference, the reagent that removes X in the step (2) is 1-5%) trifluoroacetic acid (TFA)/methylene dichloride (DCM) (volume/volume).
In another preference, remove the reaction of reagent of X in the step (2) at stirring at room 1-3 hour.
In another preference, connect L-4 on the side chain amino in the step (2), the coupling agent that uses during the 5-dihydroorotate is selected from DIC/HOBt, DIC/HOAt, PyBop/HOBt/DIEA, PyBop/HOBt/DIEA or PyBop/Cl-HOBt/DIEA; More preferably PyBop/HOBt/DIEA.
In another preference, connect L-4 on the side chain amino in the step (2), during the 5-dihydroorotate stirring at room 2-3 hour.
In another preference, with polypeptide resin cut employed lytic reagent be selected from 1. the volume ratio of trifluoroacetic acid, tri isopropyl silane, water be 90-95:2-5:2-5 or 2. the volume ratio of trifluoroacetic acid, thioanisole, methyl-phenoxide, 1，2-ethandithiol be 85-90:2-5:2-5:3-5;
And/or the reagent that removes amino protecting group Fmoc is selected from the mixing of following one or more: the 1. DMF solution of 1%-50% piperidines; 2. the DMF solution of 0.5%-10%DBU; 3. the DMF solution of 1%-10% piperazine; 4. the DMF solution of 0.5%-10%HOBt; 5. the DMF solution of 0.1%-10%DTT; 6. the DMF solution of 5%-50% thanomin; 7. the DMF solution of 5-50%TBA; 8. the DMF solution of 1%-10% U-4527; 9. the DMF solution of 3%-30%N-crassitude.
In another preference, pyrolysis time is: 60 minutes to 180 minutes, and preferred 120 minutes; Cracking temperature is 5-30 ℃, preferred 25 ℃.
In another preference, remove preferred 20% piperidines of reagent of amino protecting group Fmoc/DMF mixing solutions; The deprotection time is: 5 minutes to 30 minutes, preferred for the first time deprotection was 5 minutes, and deprotection is 10 minutes for the second time; The deprotection temperature is 5-30 ℃, preferred 25 ℃.
Accordingly, the invention provides a kind of technique of new solid phase synthesis Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2.
Description of drawings
Fig. 1 has shown that the present invention prepares the synthesis process flow diagram of fragment one.
Fig. 2 has shown that the present invention prepares the synthesis process flow diagram of fragment two.
Fig. 3 has shown that the present invention prepares the synthesis process flow diagram of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2.
Fig. 4 has shown the LC-MS collection of illustrative plates of the thick peptide of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 that the embodiment of the invention obtains.
Embodiment
The contriver is through further investigation and explore, found to use novel amido protecting amino acid Fmoc-4Aph (X)-OH, and wherein X=Mmt, Dmt, reaction conditions is gentle, reaction efficiency is high, is conducive to accomplish scale production; And find to avoid Fmoc-4Aph (Hor)-OH to contact repeatedly the alkaline condition that removes Fmoc and its side chain is changed the 6th amino acid cut-out, finally be converted into the similar thing impurity of glycolylurea.
The method for expressing that generally acknowledged in the field under the method for expressing of compound used herein, chemical group and reagent etc. was.In order conveniently to consult, below will list herein used shortenings and concrete title thereof:
| AM | Aminomethyl |
| Boc | Uncle's fourth oxygen formyl radical |
| Cbm | Formamyl |
| CTC | 2-chloro-trityl chloride |
| DCM | Methylene dichloride |
| DIC | DIC |
| DIEA | Diisopropylethylamine |
| DMF | DMF |
| Dmt | 4,4-dimethoxytrityl |
| EDT | 1，2-ethandithiol |
| Fmoc | 9-fluorenylmethyloxycarbonyl |
| HOAt | 1-hydroxyl-7-azo benzotriazole |
| PyBop | Phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl |
| HOBt | I-hydroxybenzotriazole |
| Cl-HOBt | 6-chloro-I-hydroxybenzotriazole |
| HPLC | High performance liquid chromatography |
| ipr | Sec.-propyl |
| L-Hor | L-4, the 5-dihydroorotate |
| MBHA | Toluene hydrogen polyimide resin |
| Mmt | 4-methoxyl group trityl |
| NMP | N-Methyl pyrrolidone |
| PIP | Piperidines |
| PyBop | (benzotriazole-1-base-oxygen) tripyrrole Wan Phosphonium phosphofluoric acid |
| TBTU | O-(benzotriazole-1-oxygen)-N, N, N, N-tetramethyl-urea hexafluoro borate |
| TBu or tBu | The tertiary butyl |
| TFA | Trifluoroacetic acid |
| TIS | Tri isopropyl silane |
| Trt | Trityl |
The partial structural formula that relates among the preparation method provided by the invention is as shown in the table:
As used herein, " solid phase synthesis " or " Solid-phase Polypeptide synthesizes (solid phase peptide synthesis) " is a kind of peptide synthesis technology well known in the art, includes but not limited to following method: with the protected amino acid of amino covalently bound (bonding) on solid phase carrier; Going in the presence of the protective material, the protecting group of desamidizate is received on the solid phase carrier first amino acid; Then amino be closed (protection) second amino acid whose carboxyl by activation, second amino acid that carboxyl is activated forms peptide bond with first the amino acid whose amino reaction (condensation) that is connected on solid phase carrier again, has just generated like this a dipeptides with protecting group on solid phase carrier; Repeat above-mentioned peptide bond and form reaction, peptide chain is grown, until reach needed peptide chain length from the C end to the N end; The protecting group of last deaminize, the ester bond (cutting) between hydrolysis peptide chain and the solid phase carrier obtains synthetic peptide.
As used herein, " removing protective material " or " deprotection agent " can Alternate, all refers to be connected to the chemical reagent that the amino protecting agent on the amino acid is removed; described amino protecting agent can make well known in the art; such as but not limited to, Fmoc, Boc.
As used herein, " condensing agent ", " activator ", " condensation activator " or " coupling agent " can Alternates, all be to instigate an amino acid whose amino and another amino acid whose carboxyl condensation to form the chemical reagent of peptide bond, can make well known in the art, such as but not limited to, DIC, HATU, TBTU, DIPEA.
As used herein, " cutting agent " or " cracking agent " can Alternate, all refers to the polypeptide of resin-bonded and the chemical reagent of resin isolation, can make well known in the art, such as but not limited to, contain weakly acidic solution, the HCl solution of TFA.
As used herein, " Rink Amide Linker " is the connecting arm of using during a peptide species synthesizes, and structure is shown below, and molecular formula is C
32H
29NO
7, molecular weight be 539.58, CAS number be 145069-56-3
As used herein, " Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude product " refers to that HPLC purity is at the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 product of 40%-75%.
As used herein, " room temperature " refers to 15-30 ℃, preferred 20-25 ℃.
Particularly, Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 solid phase synthesis process provided by the invention may further comprise the steps:
The first step, solid phase prepares peptide resin fragment (formula III and formula IV) respectively;
Second step forms respectively fragment one and fragment two (formula V and formula VII);
The 3rd step, fragment one and fragment two are carried out coupling, obtain suc as formula the polypeptide resin shown in the II;
In the 4th step, the polypeptide on the polypeptide resin shown in the formula II and resin isolation obtain suc as formula the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 shown in the I.
Described in the above-mentioned the first step is Fmoc-Rink Amide AM resin or Fmoc-Rink Amide mbha resin deprotection by being 0.3-0.7mmol/g with substitution degree suc as formula the peptide resin fragment shown in the III synthetic, obtain Rink Amide AM resin or Rink Amide mbha resin, then with the Fmoc-D-Ala-OH reaction, obtain Fmoc-D-Ala-Rink Amide AM resin or Fmoc-D-Ala-Rink Amide mbha resin; The method of utilizing solid phase synthesis successively coupling with the amino acid of blocking group: Fmoc-Pro-OH, Fmoc-Lys (ipr; Boc)-and OH, Fmoc-Leu-OH, Fmoc-D-4Aph (Cbm)-OH, Fmoc-4Aph (X)-OH, obtain the peptide resin (formula III) of side chain band protecting group.Wherein X is selected from Mmt or Dmt, preferred Mmt.
Described in the above-mentioned the first step is CTC-Cl resin deprotection by being 0.8-1.2mmol/g with substitution degree suc as formula the peptide resin fragment shown in the IV synthetic, obtains the CTC-Cl resin, then and Fmoc-Ser (
tBu)-OH reaction, obtain Fmoc-Ser (
tBu)-the CTC resin; The method of utilizing solid phase synthesis successively coupling obtains the peptide resin (formula IV) of side chain band protecting group with the amino acid of blocking group: Fmoc-D-3Pal-OH, Fmoc-D-Phe (4Cl)-OH, Fmoc-D-2Nal-OH.
Fragment one described in the above-mentioned second step is by sloughing the protecting group X that C holds the 6th amino-acid residue 4Aph (X) under the TFA of 1-5% suc as formula the polypeptide resin shown in the III; then connect L-4 at its side chain amino; the 5-dihydroorotate gets by removing the Fmoc group again.
The reagent of the described X of removing is the trifluoroacetic acid/dichloromethane of lower concentration, and the side chain Boc protecting group of L-Lys is not removed under this reaction conditions; Preferred 1-5% (volume/volume) TFA/DCM, stirring at room 1-3 hour.
L-4, the coupling agent that the 5-dihydroorotate uses when being connected to the 6th amino-acid residue 4Aph side chain amino is selected from DIC/HOBt, DIC/HOAtyBop/HOBt/DIEA, PyBop/HOBt/DIEA or PyBop/Cl-HOBt/DIEA, preferred PyBop/HOBt/DIEA, linked reaction was at stirring at room 2-3 hour.
Fragment two described in the above-mentioned second step is by removing Fmoc suc as formula the polypeptide resin shown in the IV, and obtaining under the TFA effect after the acetylize; The concentration of TFA is 1-5%, preferred 1%TFA/DCM(V/V) solution, the reaction times is 0.5-3.0 hour, preferred 1 hour.
Above-mentioned the 3rd step be with fragment one and fragment two by in the PyBop/HOBt/DIEA/DMF system, carrying out coupling, obtain suc as formula the polypeptide resin shown in the II.
In above-mentioned the 4th step, the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude product that obtains after the polypeptide on the polypeptide resin shown in the formula II and the resin isolation can further carry out separation and purification, more preferably can make the content of the product list impurity behind the purifying be lower than 0.3%(HPLC purity).
The coupling agent that relates in the aforesaid method is selected from DIC/HOBt, DIC/Cl-HOBt, DIC/HOAt, TBTU/HOBt/DIEA, TBTU/Cl-HOBt/DIEA, PyBop/HOBt/DIEA or PyBop/Cl-HOBt/DIEA; Reaction solvent during coupling is selected from DMF, NMP or DMF/DCM; The linked reaction temperature is room temperature, and the reaction times is for stirring 2-4 hour.
The reagent reagent that removes amino protecting group Fmoc that relates in the aforesaid method is selected from the mixing of following one or more: the 1. DMF solution of 1%-50% piperidines; 2. the DMF solution of 0.5%-10%DBU; 3. the DMF solution of 1%-10% piperazine; 4. the DMF solution of 0.5%-10%HOBt; 5. the DMF solution of 0.1%-10%DTT; 6. the DMF solution of 5%-50% thanomin; 7. the DMF solution of 5-50%TBA; 8. the DMF solution of 1%-10% U-4527; 9. the DMF solution of 3%-30%N-crassitude; Be preferably 10-50% piperidines/DMF mixing solutions or 5-10% piperazine/DMF mixing solutions.
The reagent that is used for polypeptide on the polypeptide resin and resin isolation in the aforesaid method be selected from 1. the volume ratio of trifluoroacetic acid, tri isopropyl silane, water be 90-95:2-5:2-5 or 2. the volume ratio of trifluoroacetic acid, thioanisole, methyl-phenoxide, 1，2-ethandithiol be 85-90:2-5:2-5:3-5.
In a preferred embodiment of the invention, the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude product that obtains after polypeptide on the polypeptide resin shown in the 4th step Chinese style II and the resin isolation precipitates with anhydrous isopropyl ether, pass through high-efficient liquid phase chromatogram purification, freeze-drying obtain purifying suc as formula the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 shown in the I.
In one embodiment of the invention, take Rink Amide-AM resin or Rink Amide-MBHA resin as initial vector, take the amino acid of Fmoc protection as monomer, connect successively amino acid whose method and may further comprise the steps:
1) Fmoc-D-Ala-OH is linked to each other with the amino of resin, obtain the Fmoc-D-Ala-resin;
2) adopt Fmoc solid phase synthesis strategy to connect successively six amino acid of amino acid to the of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, obtain six peptide resins that Fmoc protects
Fmoc-4Aph (X)-D-4Aph (Cbm)-Leu-Lys (ipr, Boc)-Pro-D-Ala-resin; X is Mmt or Dmt;
3) under the TFA of 1-5%, slough the protecting group X that C holds the 6th amino-acid residue 4Aph (X); then connect L-4 at its side chain amino; the 5-dihydroorotate is taken off the Fmoc group by piperidines/DMF or piperazine/DMF again and is obtained fragment one, and its peptide sequence structure is: NH
2-4Aph (Hor)-D-4Aph (Cbm)-Leu-Lys (ipr, Boc)-Pro-D-Ala-resin;
4) Fmoc-Ser (
tBu)-OH and the coupling of CTC resin obtain Fmoc-Ser (
tBu)-CTC;
5) adopt Fmoc solid phase synthesis strategy to connect successively ten amino acid of seven amino acid to the of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, obtain the tetrapeptide resin Fmoc-D-2Nal-D-Phe (4Cl) of Fmoc protection-D-3Pal-Ser (tBu)-CTC;
6) remove Fmoc, acetylize obtains AC-D-2Nal-D-Phe (4Cl)-D-3Pal-Ser (tBu)-CTC;
7) obtain AC-D-2Nal-D-Phe (4Cl)-D-3Pal-Ser (tBu)-OH under the 1%TFA effect, namely fragment two;
8) fragment one gets Ac-D-2Nal-D-Phe (4Cl)-D-3Pal-Ser (tBu)-4Aph (Hor)-D-4Aph (Cbm)-Leu-Lys (ipr, Boc)-Pro-D-Ala-resin with fragment two by solid phase method coupling in the PyBop/HOBt/DIEA/DMF system;
9) described lytic reagent is: TFA:EDT:TIS:H
2O=90:5:3:2 stirring at room 2-3 hour, adopts anhydrous isopropyl ether to precipitate thick peptide;
10) purifies and separates obtains the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 sterling, and the content of its single impurity is lower than 0.3%.
Relevant ninhydrin (Kaiser), ninhydrin test (Ninhydrin test), and monitoring method can be referring to document VIRENDER K.SARIN, et al. " Quantitative Monitoring of Solid-Phase Peptide Synthesis by the Ninhydrin Reaction " ANALYTICAL BIOCHEMISTRY117,147-157(1981), E.KAISER, et al. " Color Test for Detection of Free Terminal Amino Groups in the Solid-Phase Synthesis of Peptides " SHORT COMMUNICATIONS595-598 (Received October28,1969), with THORKILD CHRISTENSEN " A Qualitative Test for Monitoring Coupling Completeness in Solid Phase Peptide Synthesis Using Chloranil " Acta Chemica Scandinavica B33 (1979) 763-766.
The above-mentioned feature that the present invention mentions, or the feature that embodiment mentions can arbitrary combination.All features that this case specification sheets discloses can with any composition forms and usefulness, each feature that discloses in the specification sheets can anyly provide the alternative characteristics of identical, impartial or similar purpose to replace.Therefore except special instruction is arranged, the feature that discloses only is the general example of equalization or similar features.
Major advantage of the present invention is:
1, the present invention has adopted the Fmoc synthesis strategy, has avoided hydrofluoric use in the Boc synthesis strategy, and is more friendly to environment.
2, to hold the 6th amino acid to adopt Fmoc-4Aph (X)-OH be raw material to C of the present invention, and wherein X is Mmt or Dmt, and cut off at this place, avoided repeatedly contacting of L-Hor and alkali, the possibility of the similar thing impurity of formation glycolylurea after having reduced it and resetting.
3, to hold the 6th amino acid to adopt Fmoc-4Aph (X)-OH be raw material to C of the present invention, and wherein X is Mmt or Dmt, and preferred Mmt removes the reaction conditions milder of Mmt or Dmt, operates simplyr, is easy to large-scale production.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is usually according to normal condition or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise all percentage ratio, ratio, ratio or umber by weight.
Unit in the percent weight in volume among the present invention is well-known to those skilled in the art, for example refers to the weight of solute in 100 milliliters solution.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The usefulness that better implementation method described in the literary composition and material only present a demonstration.
Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 method for detecting purity of the present invention is:
Pillar: Kromasil C18,5um, 4.6*250mm
Wavelength: 214nm
Flow velocity: 1mL/min
Moving phase: A:H
2O+0.1%TFA
B：ACN+0.1%TFA
Gradient: A B
The purification process of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 of the present invention is:
Pillar: Kromasil C18,10 μ m, 50*250mm
Wavelength: 214nm
Flow velocity: 100mL/min
Moving phase: A:H
2O+0.1%TFA
B：ACN+0.1%TFA
Gradient: A B
Synthesizing of Fmoc-D-Ala-AM resin
Rink Amide-AM resin (5mmol, substitution degree 0.5mmol/g) is added solid phase reactor, add 100ml DMF swelling 30min, with twice of DMF washing.Add 100ml20%PIP/DMF solution and take off Fmoc protection 10min, drain, add again 100ml20%PIP/DMF solution and take off Fmoc protection 20min, DMF washing 3 times, DCM washing 2 times, DMF washing 3 times.4.67g Fmoc-D-Ala-OH(15.0mmol, 3.0eq.) and 3.04g HOBt(22.5mmol, 4.5eq.) be dissolved among the 80ml DMF, add 4.7ml DIC(30.0mmol in 0-5 ℃, 6.0eq.), activate in advance 2-5min, add in the solid phase reactor, drum nitrogen reaction 2-3h, triketohydrindene hydrate detects and is negative.Drain, DMF washing 3 times, DCM washing 3 times can obtain the Fmoc-D-Ala-AM resin after the drying, and its substitution degree is 0.42mmol/g after testing.
Synthesizing of Fmoc-D-Ala-MBHA resin
Rink Amide-MBHA resin (5mmol, substitution degree 0.8mmol/g) is added solid phase reactor, add 100ml DMF swelling 30min, with twice of DMF washing.Add 100ml20%PIP/DMF solution and take off Fmoc protection 10min, drain, add again 100ml20%PIP/DMF solution and take off Fmoc protection 20min, DMF washing 3 times, DCM washing 2 times, DMF washing 2 times.4.67g Fmoc-D-Ala-OH and 3.04gHOBt are dissolved among the 80ml DMF, in 0-5 ℃ of adding 4.7ml DIC, activate in advance 2-5min, add in the solid phase reactor, and drum nitrogen reaction 2-3h, triketohydrindene hydrate detects and is negative.Drain, DMF washing 3 times, DCM washing 3 times can obtain the Fmoc-D-Ala-MBHA resin after the drying, and its substitution degree is 0.58mmol/g after testing.
Embodiment 3
NH
2Synthesizing of-4Aph (Mmt)-D-4Aph (Cbm)-Leu-Lys (ipr, Boc)-Pro-D-Ala-MBHA resin
Fmoc-D-Ala-MBHA (5mmol) resin is added solid phase reactor, and DMF washing 2 times adds 80mlDMF swelling 30min again; drain, add 20%PIP/DMF and take off twice (time is respectively 10min+15min) of Fmoc protection, DMF washing 3 times; DCM washing 2 times, DMF washing 3 times.
5.06g Fmoc-Pro-OH and 3.04gHOBt are dissolved among the 60ml DMF, in 0-5 ℃ of adding 4.7ml DIC, activate in advance 2-5min, add in the solid phase reactor, and drum nitrogen reaction 2-3h, triketohydrindene hydrate detects and is negative.Drain DMF washing 3 times.Add 20%PIP/DMF and take off twice (time is respectively 10min+15min) of Fmoc protection, DMF washing 3 times, DCM washing 2 times, DMF washing 3 times.
Repeat above step; connect successively Fmoc-Lys (ipr; Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4Aph (Cbm)-OH; add 20%PIP/DMF and take off twice (time is respectively 10min+15min) of Fmoc protection; DMF washing 3 times; DCM washing 2 times, the DMF washing obtains Fmoc--4Aph (Mmt)-D-4Aph (Cbm)-Leu-Lys (ipr, Boc)-Pro-D-Ala-MBHA resin 3 times.
Embodiment 4
NH
2Synthesizing of-4Aph (Hor)-D-4Aph (Cbm)-Leu-Lys (ipr, Boc)-Pro-D-Ala-MBHA resin
Add 1%TFA/DCM solution, drum nitrogen reaction 2-3h, triketohydrindene hydrate detects and is negative.Drain DCM washing 3 times, DMF washing 3 times.
2.37g L-4,, 5 dihydroorotates and 2.03g HOBt are dissolved among the 100ml DMF, in 0-5 ℃ of adding 10.4g PyBop, add in the solid phase reactor.DIPEA is added dropwise in the reactor, and maintenance system pH value is about 7.Reaction is roused nitrogen gas stirring after 3 hours in room temperature,, triketohydrindene hydrate detects and is negative.Drain DMF washing 3 times.Add 20%PIP/DMF and take off twice (time is respectively 10min+15min) of Fmoc protection, DMF washing 3 times, DCM washing 2 times, DMF washing 3 times can obtain fragment one, and its peptide order structure is: NH
2-4Aph (Hor)-D-4Aph (Cbm)-Leu-Lys (ipr, Boc)-Pro-D-Ala-MBHA resin
Embodiment 5
Synthesizing of Fmoc-D-2Nal-D-Phe (4Cl)-D-3Pal-Ser (tBu)-CTC resin
CTC-Cl resin (5mmol, substitution degree 1.0mmol/g) is added solid phase reactor, add 100mlDMF swelling 30min, with twice of DMF washing.Add Fmoc-Ser (tBu)-OH(1.0eq.), then add DIEA(1.5eq.), stirring at room 3 hours, resin is used respectively DMF after filtering, and DCM, methyl alcohol wash 3 times, vacuum-drying.
3.88g Fmoc-D-3-Pal-OH and 3.04gHOBt are dissolved among the 60ml DMF, in 0-5 ℃ of adding 4.7ml DIC, activate in advance 2-5min, add in the solid phase reactor, and drum nitrogen reaction 2-3h, triketohydrindene hydrate detects and is negative.Drain DMF washing 3 times.Add 20%PIP/DMF and take off twice (time is respectively 10min+15min) of Fmoc protection, DMF washing 3 times, DCM washing 2 times, DMF washing 3 times.Repeat the above peptide that connects and operate, obtain Fmoc-D-3Pal-OH-D-Phe (4Cl)-OH-D-2Nal-Ser (tBu)-CTC resin.
Embodiment 6
Ac-D-2Nal-D-Phe (4Cl)-D-3Pal-Ser (tBu)-OH's is synthetic
Add 20%PIP/DMF and take off twice (time is respectively 10min+15min) of Fmoc protection, DMF washing 3 times, DCM washing 2 times, DMF washing 3 times.Add solid phase reactor after adding 20g diacetyl oxide and 16g pyridine mixing, stir 2-3h, triketohydrindene hydrate detects and is negative.Drain, DMF washing 5 times, obtain Ac-D-3Pal-OH-D-Phe (4Cl)-OH-D-2Nal-Ser (tBu)-CTC resin, then process obtaining fragment two: Ac-D-3Pal-OH-D-Phe (4Cl)-OH-D-2Nal-Ser (tBu)-OH through 1%TFA.
Embodiment 7
Synthesizing of AC-D-2Nal-D-Phe (4Cl)-D-3Pal-Ser (tBu)-4Aph (Hor)-D-4Aph (Cbm)-Leu-Lys (ipr, Boc)-Pro-D-Ala-MBHA resin
7.30g Ac-D-3Pal-OH-D-Phe (4Cl)-OH-D-2Nal-Ser (tBu)-OH and 2.03g HOBt are dissolved among the 100ml DMF, in 0-5 ℃ of adding 10.4g PyBop, add and are equipped with in the solid phase reactor of fragment one.DIPEA is added dropwise in the reactor, and maintenance system pH value is about 7.After 3 hours, triketohydrindene hydrate detects and is negative at room temperature drum nitrogen gas stirring in reaction.Drain DMF washing 3 times.
Embodiment 8
The cracking of peptide resin
The 20.18g peptide resin adds 250ml0-5 ℃ lytic reagent (TFA:TIS:EDT:H among the embodiment 7
2O=90:3:5:2) in, stirring at room reaction 3h filters, and joins in the filtrate in 2.5L0-5 ℃ the anhydrous diethyl ether, and is centrifugal, obtains the thick peptide of 9.02g after the washing drying.The LC-MS collection of illustrative plates of thick peptide is seen Fig. 4.
Embodiment 9
The purifying of thick peptide
Add approximately 10 times of acetonitrile/water solution dissolving crude products, filter, utilize two-step purifying scheme purified product.Use the packed column of reverse C18, in the first step purifying, the moving phase system adopts the ethanol of the 0.1%TFA aqueous solution and 99.9%, collects purity and is higher than 99% cut; In the purifying, the moving phase system comprises 1% acetic acid aqueous solution, 99.9% ethanol and 0.5M ammonium acetate solution for the second time, collects purity and is higher than 99.8% cut.Concentrated, freeze-drying can obtain the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 solid of puffy.
The above only is preferred embodiment of the present invention, be not to limit essence technology contents scope of the present invention, essence technology contents of the present invention is broadly to be defined in the claim scope of application, any technology entity or method that other people finish, if defined identical with the claim scope of application, also or a kind of change of equivalence, all will be regarded as being covered by among this claim scope.
Claims (8)
1. solid phase synthesis preparation method thereof suc as formula the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 shown in the I; Fmoc-amino acid is connected with resin; obtain Fmoc-amino acid-resin; adopt the step of method through amino acid is held in order successively connection and the Fmoc group is removed to N from the C end of solid phase synthesis; obtain the polypeptide resin of Fmoc protection; it is characterized in that, described method comprises step:
(1) forms respectively suc as formula the polypeptide resin shown in III and the formula IV;
(2) will mix suc as formula the trifluoracetic acid (TFA) of the polypeptide resin shown in the III and 1-5%, after sloughing the protecting group X that C holds the 6th amino-acid residue, connect L-4 at its side chain amino, the 5-dihydroorotate removes the Fmoc group and obtains suc as formula the polypeptide resin shown in the V;
To remove Fmoc group, acetylize suc as formula the polypeptide resin shown in the IV obtains obtaining suc as formula the fragment shown in the VII suc as formula cutting behind the polypeptide resin shown in the VI;
(3) will carry out coupling by solid phase method suc as formula the polypeptide resin shown in the V with suc as formula the fragment shown in the VII, obtain suc as formula the polypeptide resin shown in the II; With
(4) polypeptide and the resin isolation on the polypeptide resin shown in the formula II obtains suc as formula the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 shown in the I;
Wherein X is selected from Mmt or Dmt; Preferred Mmt.
2. preparation method as claimed in claim 1 is characterized in that, is selected from Rink Amid-MBHA resin or Rink Amide-AM resin suc as formula the resin in the polypeptide resin shown in the III, and substitution degree is 0.3-0.7mmol/g; Substitution degree suc as formula the resin CTC in the polypeptide resin shown in the IV is 0.8-1.2.
3. preparation method as claimed in claim 1; it is characterized in that; the amino acid of the protection of formation during suc as formula the polypeptide resin shown in the III is respectively Fmoc-D-Ala-OH, Fmoc-Pro-OH, Fmoc-Lys (ipr, Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4Aph (Cbm)-OH, Fmoc-4Aph (X)-OH.
4. preparation method as claimed in claim 1 is characterized in that, the amino acid of the protection when forming suc as formula the polypeptide resin shown in the IV be respectively Fmoc-Ser (
tBu)-OH, Fmoc-D-3Pal-OH, Fmoc-D-Phe (4Cl)-OH, Fmoc-D-2Nal-OH.
5. preparation method as claimed in claim 1, it is characterized in that, the coupling agent of use is selected from DIC/HOBt, DIC/Cl-HOBt, DIC/HOAt, TBTU/HOBt/DIEA, TBTU/Cl-HOBt/DIEA, PyBop/HOBt/DIEA or PyBop/Cl-HOBt/DIEA.
6. preparation method as claimed in claim 1 is characterized in that, the reagent that removes X in the step (2) is 1-5%) trifluoroacetic acid (TFA)/methylene dichloride (DCM) (volume/volume).
7. preparation method as claimed in claim 1, it is characterized in that, connect L-4 on the side chain amino in the step (2), the coupling agent that uses during the 5-dihydroorotate is selected from DIC/HOBt, DIC/HOAt, PyBop/HOBt/DIEA, PyBop/HOBt/DIEA or PyBop/Cl-HOBt/DIEA; Preferred PyBop/HOBt/DIEA.
8. such as each described preparation method of claim 1-7, it is characterized in that, with polypeptide resin cut employed lytic reagent be selected from 1. the volume ratio of trifluoroacetic acid, tri isopropyl silane, water be 90-95:2-5:2-5 or 2. the volume ratio of trifluoroacetic acid, thioanisole, methyl-phenoxide, 1，2-ethandithiol be 85-90:2-5:2-5:3-5;
And/or the reagent that removes amino protecting group Fmoc is selected from the mixing of following one or more: the 1. DMF solution of 1%-50% piperidines; 2. the DMF solution of 0.5%-10%DBU; 3. the DMF solution of 1%-10% piperazine; 4. the DMF solution of 0.5%-10%HOBt; 5. the DMF solution of 0.1%-10%DTT; 6. the DMF solution of 5%-50% thanomin; 7. the DMF solution of 5-50%TBA; 8. the DMF solution of 1%-10% U-4527; 9. the DMF solution of 3%-30%N-crassitude.
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