CN106928313B - Synthesis method of C-terminal modified peptide - Google Patents
Synthesis method of C-terminal modified peptide Download PDFInfo
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- CN106928313B CN106928313B CN201511027301.4A CN201511027301A CN106928313B CN 106928313 B CN106928313 B CN 106928313B CN 201511027301 A CN201511027301 A CN 201511027301A CN 106928313 B CN106928313 B CN 106928313B
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
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- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
- C07K1/061—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups
- C07K1/062—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups for alpha- or omega-carboxy functions
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- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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Abstract
The invention provides a method for synthesizing C-terminal modified peptide, which is characterized by comprising the following steps: 1) coupling one end amino group of the diamino compound to the solid-phase synthetic resin; 2) adopting Fmoc solid-phase peptide synthesis strategy to couple amino acid to the amino group at the other end of the diamino compound in sequence to obtain fully-protected polypeptide resin; 3) cleaving the fully protected polypeptide from the resin to obtain a fully protected polypeptide; 4) removing a protecting group from the fully protected polypeptide to obtain the target C-terminal modified peptide, or 5) coupling the fully protected polypeptide with a carboxyl-containing modifying group to obtain the target C-terminal modified peptide. The method has the advantages of simple operation, energy conservation, environmental protection, yield improvement and the like.
Description
Technical Field
A synthetic method for preparing C-terminal modified peptide.
Background
Polypeptide modification is an important means for changing the main chain structure and side chain groups of a peptide chain, a great deal of literature reports are available on changing the physicochemical properties of peptide compounds and playing more and more important roles in effectively utilizing the peptide compounds in vivo, and the modified polypeptide drug can obviously reduce immunogenicity, reduce toxic and side effects, increase water solubility, prolong the action time in vivo, change the biological distribution condition of the polypeptide drug and the like, thereby obviously improving the curative effect of the drug.
At present, chemical modification of the C-terminal of the polypeptide is becoming a research hotspot, and introduction of some small molecule drugs at the C-terminal is becoming a novel research direction. For the C-terminal modification of polypeptide, the modification of amino groups can be completed by directly carrying out amido bond reaction with carboxyl at the C-terminal of a peptide chain in a liquid phase; for the modification of carboxyl functional group at C-terminal, a diamido Linker is generally required to be introduced between the C-terminal carboxyl of the peptide chain and the modified carboxyl, and the method undoubtedly increases the chemical synthesis difficulty of the peptide, because the amide bond reaction is generally carried out twice in liquid phase, the post-treatment difficulty of the synthesis is greatly increased, and the yield of the target peptide is greatly reduced.
At present, aiming at C-terminal carboxyl modification of polypeptide, the main synthesis technology is to complete coupling of peptide sequence on 2-Chlorotrityl Chloride Resin by Fmoc solid-phase peptide synthesis strategy, wherein amino at N-terminal adopts Boc protection, then cracking to obtain fully-protected peptide, then introducing a diammine Linker (generally ethylenediamine, butanediamine, hexanediamine and octanediamine …) at C-terminal of peptide chain in liquid phase, and introducing modified carboxyl group in liquid phase again after post-treatment. GHWDFRQWWQPSGGGS-hexamethylenediamine-Biotin is taken as an example to illustrate the prior synthesis technical scheme, and the scheme is shown in figure 1.
Disclosure of Invention
In order to overcome various defects of the synthesis of the C-terminal modified peptide, the invention aims to introduce a novel method for directly coupling resin with diamino Linker. For peptides (such as epristeride) whose C-terminal is modified with diamino, the synthesis of the target product can be completed directly on the solid phase, as shown in FIG. 2. The method of the invention accomplishes the coupling of the diamine compound and the solid phase carrier and the loading amount of the first amino acid residue on the solid phase carrier by controlling the reaction conditions. Meanwhile, in the process of coupling the diamine compound and the first amino acid residue, the generation of byproducts is avoided by a two-step blocking method, and the technical barrier that the bifunctional compound is difficult to be uniformly coupled to the solid phase carrier in the prior art is solved. The method has the advantages of simple operation, energy conservation, environmental protection, yield improvement and the like.
One aspect of the present invention provides a method for synthesizing a C-terminal modified peptide, comprising the steps of:
1) coupling one end amino group of the diamino compound to the solid-phase synthetic resin;
2) adopting Fmoc solid-phase peptide synthesis strategy to couple amino acid to the amino group at the other end of the diamino compound in sequence to obtain fully-protected polypeptide resin;
3) cleaving the fully protected polypeptide from the resin to obtain a fully protected polypeptide;
4) deprotecting the fully protected polypeptide to obtain the desired C-terminally modified peptide, or
4) Coupling the fully protected polypeptide with a carboxyl-containing modification group to obtain the target C-terminal modified peptide.
Further, the diamino compound is selected from linear symmetrical diamino compounds, branched symmetrical diamino compounds or diamino compounds with symmetrical spatial structure, preferably ethylenediamine, propylenediamine, butylenediamine, pentylenediamine, hexylenediamine, heptylenediamine, octylenediamine, dodecylenediamine, dodecylamine,
Further, the solid phase synthetic Resin is selected from 2-Chlorotrityl Chloride Resin, preferably, the substitution degree of the solid phase synthetic Resin is in the range of 0.1-1.2 mmol/g, more preferably 0.2-0.8 mmol/g, and most preferably 0.3-0.5 mmol/g.
Further, the coupling system used in the Fmoc solid-phase peptide synthesis strategy in step 2) is DIC + A or B + A + C, wherein A is HOBt or HOAt, B is HBTU, HATU, TBTU or PyBOP, and C is DIPEA or TMP.
Further, the cleavage reagent used in the cleavage step of step 3) is a combination of TFA, TIS, EDT, H2O, the cleavage time is 1.5-3.5 hours, and the preferred volume ratio of the cleavage reagent is TFA: and (3) TIS: EDT (electro-thermal transfer coating): H2O-85-95: 2-5:2-5:1-5,
further, step 4) is followed by a purification step, preferably by HPLC.
Further, the using method of the step 1) comprises the steps of weighing solid-phase synthetic resin, placing the solid-phase synthetic resin in a solid-phase reaction column, adding DMF (dimethyl formamide), and carrying out bubbling swelling by nitrogen; weighing a diamino compound, dissolving the diamino compound with DMF (dimethyl formamide), adding DIPEA into a solid-phase reaction column, adding methanol and DIPEA after complete reaction, mixing and sealing for 10-60 minutes, washing with DCM (diethyl formamide), and draining the resin after methanol shrinkage to obtain the diamino compound coupled solid-phase synthetic resin.
Further wherein the step of coupling the first amino acid to the other amino group of the diamino compound in step 2) is: adding DMF into the diamino compound coupled solid-phase synthetic resin obtained in the step 1), and carrying out nitrogen bubbling for swelling; activating the amino acid protected by Fmoc by using a coupling system, adding the activated amino acid protected by Fmoc into a reaction column for reaction, then adding acetic anhydride and pyridine, mixing and sealing to obtain the amino acid diamino compound coupled solid-phase synthetic resin protected by Fmoc.
Further, wherein the amino acid sequence coupled sequentially to the other amino group of the amino compound is selected from the group consisting of polypeptides comprising 1-20 amino acids, preferably 5-15 amino acids.
Further, the carboxyl-containing modifying group is selected from Biotin, fluorescein, carnitine, formic acid, acetic acid, palmitic acid, stearic acid, cholic acid, carboxyl-containing small molecule drugs and pharmacodynamic functional groups.
Drawings
FIG. 1 is a flow chart of the conventional synthesis of polypeptide C-terminal modified Biotin;
FIG. 2 is a flow chart of the synthesis of polypeptide C-terminal modified Biotin of the present invention.
Detailed Description
2 2 8Example 1 NH-Preparation of (CH) -NH-2-Chlorotrityl Chloride Resin
Weighing 250 g of 2-Chlorotrityl Chloride Resin with the substitution degree of 0.8mmol/g into a solid phase reaction column, adding DMF, and carrying out bubbling swelling for 60 minutes by nitrogen; weighing 57.7 g (400mmol) of octanediamine, dissolving with DMF, adding 90.0ml DIPEA (500mmol) at 0 ℃ in ice water bath, adding into a reaction column, reacting for 1 hour, adding 200ml methanol and 200ml DIPEA, mixing and sealing for 0.5 hour, washing with DCM for three times, contracting with methanol, and draining off the resin to obtain NH2-(CH2)8-NH-2-Chlorotrityl Chloride Resin。
2 8Example 2 preparation of Fmoc-Phe-NH- (CH) -NH-2-Chlorotrityl Chloride Resin
NH obtained in example 1 was weighed2-(CH2)8Adding 100 grams of-NH-2-Chlorotrityl Chloride Resin into a solid phase reaction column, adding DMF, and carrying out bubbling with nitrogen for swelling for 60 minutes; 23.22 g (60mmol) of Fmoc-Phe-OH, 9.72 g (72mmol) of HOBt and 33.8 g (60mmol) of HBTU are weighed, dissolved by DMF, 15.6ml of DIPEA (72mmol) is added in ice-water bath at 0 ℃, activated for 5 minutes and added into a reaction column, after 2 hours of reaction, 70ml of acetic anhydride and 60ml of pyridine are added, mixed and sealed for 24 hours, washed three times by DCM, and the resin is drained after methanol contraction to obtain Fmoc-Phe-NH- (CH)2)8-NH-2-Chlorotrityl Chloride Resin, 124g of Resin with a degree of substitution of 0.51mmol/g was examined.
Example 3 preparation of an Epilatide peptide resin
The structure of the epriside is as follows: H-Met (O)2)-Glu-His-Phe-D-Lys-Phe-NH-(CH2)8-NH2。
Fmoc-Phe-NH- (CH) with a degree of substitution of 0.51mmol/g as in example 2 was weighed2)898 g (50mmol) of-NH-2-Chlorotrityl Chloride Resin is put into a solid phase reaction column, DMF is added, and nitrogen is bubbled for swelling for 60 minutes; the Fmoc protecting group was removed with DBLK and washed 6 times with DMF. Fmoc-D-Lys (Boc) -OH 468 g (100mmol) and HOBt 16.3g (120mmol) were weighed, dissolved in DMF, 15.1 g DIC (120mmol) was added to a 0 ℃ ice water bath, activated for 5 minutes, and added to a reaction column, and reacted for 2After hours, the resin was washed three times with DMF, the Fmoc protecting group was removed with DBLK, washed 6 times with DMF and 3 times with DCM. The above coupling procedure was repeated to sequentially couple Fmoc-Phe-OH, Fmoc-His (Trt) -OH, Fmoc-Glu (OtBu) -OH, Boc-Met (O) according to the peptide sequence2) -OH. After the reaction, the reaction mixture was shrunk with methanol and dried in vacuo to obtain 142g of an eprisidine peptide resin.
Example 4 preparation of crude Epilatide peptide
142g of the peptide resin obtained in example 3 was charged into a 2000ml single-neck flask, and lysate 1400ml of TFA was prepared: and (3) TIS: EDT (electro-thermal transfer coating): h2O91: 3:3:3 (vol.%), the lysate was added to a flask, the reaction was carried out at room temperature for 2.5 hours, the resin was filtered off, the resin was washed with 50ml tfa, the filtrates were combined and added to 20000ml of chilled dry ether to precipitate a white solid, which was centrifuged, washed with dry ether and dried under vacuum to give 49.6g of a white solid, 99.8% yield and 91.4% purity by HPLC.
Example 5 preparation of Ebidopeptide Fine peptide
49.6g of crude peptide of the epristeride obtained in example 4 were taken, and the obtained crude peptide was purified by a Waters 2454RP-HPLC system at a wavelength of 220nm, and the chromatographic column was a 100X 500mm reversed phase C18 column, mobile phase: phase A: 0.3% TFA/acetonitrile (v/v); phase B: acetonitrile, gradient: b%: 38% -68%, flow rate: and collecting target peak fractions at 6 ml/min, performing rotary evaporation concentration, and freeze-drying to obtain 42.5g of the epristeride refined peptide, wherein the HPLC purity is 99.34%, and the total yield is 85.4%.
Example 6 preparation of Fmoc-Phe-2-Chlorotrityl Chloride Resin
Weighing 100 g of 2-Chlorotrityl Chloride Resin with the substitution degree of 1.0mmol/g into a solid phase reaction column, adding DMF, and carrying out bubbling swelling for 60 minutes by nitrogen; Fmoc-Phe-OH 23.22 g (60mmol), HOBt 9.72 g (72mmol) and HBTU 33.8 g (600mmol) were weighed, dissolved in DMF, 15.6ml DIPEA (72mmol) was added in an ice water bath at 0 deg.C, activated for 5 minutes, added to the reaction column, reacted for 2 hours, 100ml methanol and 100ml DIPEA were added, mixed and blocked for 0.5h, washed three times with DCM, and after methanol contraction, the Resin was drained to give Fmoc-Phe-2-Chlorotrityl Chloride Resin, 119g of which was examined for substitution of 0.48 mmol/g.
2Example 7 Boc-Met (O) -Glu (OtBu) -His (Trt) -Phe-D-Lys (Boc) -Phe-2-
Preparation of Chlorotrityl Chloride Resin
Weighing 104 g (50mmol) of Fmoc-Phe-2-Chlorotrityl Chloride Resin with the substitution degree of 0.48mmol/g in example 6 into a solid phase reaction column, adding DMF, and carrying out bubbling and swelling with nitrogen for 60 minutes; the Fmoc protecting group was removed with DBLK and washed 6 times with DMF. Fmoc-D-Lys (Boc) -OH 468 g (100mmol), HOBt 16.3g (120mmol) were weighed, dissolved in DMF, 15.1 g DIC (120mmol) were added in an ice-water bath at 0 deg.C, activated for 5 min, the reaction column was added, after 2h of reaction, the resin was washed three times with DMF, the Fmoc protecting group was removed with DBLK, washed 6 times with DMF and washed 3 times with DCM. The above coupling procedure was repeated to sequentially couple Fmoc-Phe-OH, Fmoc-His (Trt) -OH, Fmoc-Glu (OtBu) -OH, Boc-Met (O) according to the peptide sequence2) -OH. After the reaction is finished, the Boc-Met (O) is obtained after methanol contraction and vacuum drying2)-Glu(OtBu)-His(Trt)-Phe-D-Lys(Boc)-Phe-2-Chlorotrityl Chloride Resin 138g。
2Example 8 preparation of Boc-Met (O) -Glu (OtBu) -His (Trt) -Phe-D-Lys (Boc) -Phe-OH
138 g of the peptide resin obtained in example 7 was placed in a 2000ml single neck flask, 1500ml of a DCM solution prepared with 0.1% TFA was placed in the flask, the reaction was carried out at room temperature for 2.0 hours, the resin was filtered off, and the solvent was rotary evaporated to give 68g of white fully protected crude peptide.
Example 9 preparation of crude Epilatide fully protected peptide
In a 500ml single-neck flask, 14.4g (100mmol) of octanediamine was charged, dissolved in 50ml of DMF, and HOBt 16.3g (120mmol) and DIC 15.1 g (120mmol) were added. 68g of the fully protected crude peptide obtained in example 8 were dissolved in 200ml of DCM, slowly added dropwise to the octanediamine solution, and after the addition was completed, the reaction was continued for 2 hours to complete the reaction. 100ml of water was added to the reaction solution, the organic phase (DCM) was collected by extraction and rotary evaporation gave 36g of crude white fully protected peptide of Ebidopeptide.
Example 10 preparation of crude Epilatide peptide
To the fully protected crude efletiritide of example 9 was added a pre-configured lysate TFA: and (3) TIS: EDT (electro-thermal transfer coating): h280ml of O91: 3:3:3 (volume ratio), reacting at room temperature for 2.5 hours, adding into 10000ml of frozen anhydrous ether, precipitating white solid, centrifuging, washing the solid with anhydrous ether, and drying in vacuum to obtain 27.5g of white solid, wherein the yield is 55.28 percent and the HPLC purity is 84.6 percent.
Example 11 preparation of Ebidopeptide Fine peptide
27.5g of crude peptide of the epristeride obtained in example 10 were taken, and the obtained crude peptide was purified by a Waters 2454RP-HPLC system at a wavelength of 220nm, and the chromatographic column was a 100X 500mm reversed phase C18 column, mobile phase: phase A: 0.3% TFA/acetonitrile (v/v); phase B: acetonitrile, gradient: b%: 38% -68%, flow rate: and collecting target peak fractions at 6 ml/min, performing rotary evaporation concentration, and freeze-drying to obtain 18.56g of the epristeride refined peptide, wherein the HPLC purity is 99.01%, and the total yield is 37.3%.
The methods of examples 1-5 (invention) and examples 6-11 (prior art) produced epirubicin in the same dosage with a total yield of approximately 3-fold difference, and for 7-peptide conjugates the total yield of refined peptide was typically around 60%, whereas the inventors surprisingly found that the effect far exceeded the expected yield by over 80%.
2 2 2Example 12 preparation of NH- (CH) -NH-2-Chlorotrityl Chloride Resin
Weighing 200 g of 2-Chlorotrityl Chloride Resin with the substitution degree of 1.0mmol/g into a solid phase reaction column, adding DMF, and carrying out bubbling swelling for 60 minutes by nitrogen; weighing 24.1 g (400mmol) of ethylenediamine, dissolving with DMF, adding 90.0ml of DIPEA (500mmol) at 0 ℃ in ice-water bath, adding into a reaction column, reacting for 1 hour, adding 200ml of methanol and 200ml of DIPEA, mixing and sealing for 0.5h, washing with DCM for three times, contracting with methanol, and draining off resin to obtain NH2-(CH2)2-NH-2-Chlorotrityl Chloride Resin。
2 2Example 13 preparation of Fmoc-Leu-NH- (CH) -NH-2-Chlorotrityl Chloride Resin
NH obtained in example 12 was weighed2-(CH2)2Adding 100 grams of-NH-2-Chlorotrityl Chloride Resin into a solid phase reaction column, adding DMF, and carrying out bubbling with nitrogen for swelling for 60 minutes; weighing 21.2 g (60mmol) of Fmoc-Leu-OH and 9.72 g (72mmol) of HOBt, dissolving with DMF, adding 16.4ml DIC (72mmol) in ice-water bath at 0 ℃, activating for 5 min, adding into a reaction column, reacting for 2h, adding 70ml acetic anhydride and 60ml pyridine, mixing and sealing for 24h, washing with DCM for three times, contracting with methanol, and draining off the resin to obtain Fmoc-Leu-NH- (CH)2)2-NH-2-Chlorotrityl Chloride Resin, detecting 118g of Resin with a degree of substitution of 0.53 mmol/g.
2 2Example 14 Boc-Phe-Phe-Gly-Ala-Ser (tBu) -Cys (Trt) -Leu-NH- (CH) -NH-2-
Preparation of Chlorotrityl Chloride Resin
The structure for preparing the target compound is as follows: H-Phe-Phe-Gly-Ala-Ser-Cys-Leu-NH- (CH)2)2-NH-L-Carnitine (L-Carnitine: L-D-base:
) (ii) a The compound is used for reducing weight, and the connecting peptide sequence improves the metabolic stability.
Fmoc-Leu-NH- (CH) with substitution 0.53mmol/g in example 13 was weighed2)2Adding 94 g (50mmol) of-NH-2-Chlorotrityl Chloride Resin into a solid phase reaction column, adding DMF, and carrying out bubbling swelling on nitrogen for 60 minutes; the Fmoc protecting group was removed with DBLK and washed 6 times with DMF. Fmoc-Cys (Trt) -OH 586 g (100mmol), HOBt 16.3g (120mmol), HBTU 38 g (100mmol) were weighed, dissolved in DMF, and 16.0 g DIPEA (120mmol) was added in an ice-water bath at 0 deg.C, activated for 5 min, added to the reaction column, and after 2 hours of reaction, the resin was washed three times with DMF, the Fmoc protecting group was removed with DBLK, washed 6 times with DMF, and washed 3 times with DCM. The above coupling procedure was repeated, Fmoc-Ser (tBu) -OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Boc-Phe-OH were coupled in sequence according to the peptide order. After the reaction is finished, the peptide is obtained after the reaction is shrunk by methanol and dried in vacuum165g of a resin.
2 2 2Example 15 preparation of Boc-Phe-Phe-Gly-Ala-Ser (tBu) -Cys (Trt) -Leu-NH- (CH) -NH
165g of the peptide resin obtained in example 14 was charged into a 2000ml single-neck flask, 1800ml of DCM solution prepared with 0.1% TFA was charged into the flask, and reacted at room temperature for 2.0 hours, after filtering off the resin, the solvent was rotary evaporated to give 64g of crude white fully protected peptide.
2 2Example 16 Boc-Phe-Phe-Gly-Ala-Ser (tBu) -Cys (Trt) -Leu-NH- (CH) -NH-L-
Preparation of Carnitine
In a 500ml single neck flask, 16.1g (100mmol) of L-Carnitine was charged, dissolved in 50ml of DMF, and 16.3g (120mmol) of HOBt and 15.1 g of DIC (120mmol) were added. 64g of the fully protected crude peptide obtained in example 15 was dissolved in 200ml of DCM, slowly added dropwise to the L-Carnitine solution, and after the dropwise addition was completed, the reaction was continued for 2h, and the reaction was completed. 100ml of water was added to the reaction mixture, and the organic phase (DCM) was collected by extraction and rotary evaporated to give Boc-Phe-Phe-Gly-Ala-Ser (tBu) -Cys (Trt) -Leu-NH- (CH)2)241g of crude L-Carnitine.
2 2Example 17 preparation of H-Phe-Phe-Gly-Ala-Ser-Cys-Leu-NH- (CH) -NH-L-Carnitine crude
Preparation of
To the crude product from example 16 was added the pre-prepared lysate TFA: and (3) TIS: EDT (electro-thermal transfer coating): h280ml of O-85: 5:5:5 (volume ratio), reacting at room temperature for 2.0 hours, adding into 10000ml of frozen anhydrous ether, precipitating white solid, centrifuging, washing the solid with anhydrous ether, and drying in vacuum to obtain 32.4g of white solid, wherein the yield is 70.1 percent, and the HPLC purity is 80.6 percent.
2 2Example 18 purification of H-Phe-Phe-Gly-Ala-Ser-Cys-Leu-NH- (CH) -NH-L-Carnitine
32.4g of the crude product obtained in example 17 are taken, and the product is purified by a Waters 2454RP-HPLC system at a wavelength of 220nm on a 100X 500mm reverse phase C18 column, mobile phase: phase A: 0.3% TFA/acetonitrile (v/v); phase B: acetonitrile, gradient: b%: 38% -68%, flow rate: and collecting target peak fractions at 6 ml/min, performing rotary evaporation concentration, and freeze-drying to obtain 25.2g of target refined peptide, wherein the HPLC purity is 99.70%, and the total yield is 54.3%.
EXAMPLE 19 preparation of Fmoc-Leu-2-Chlorotrityl Chloride Resin
Weighing 100 g of 2-Chlorotrityl Chloride Resin with the substitution degree of 1.0mmol/g into a solid phase reaction column, adding DMF, and carrying out bubbling swelling for 60 minutes by nitrogen; 21.2 g (60mmol) of Fmoc-Leu-OH, 9.72 g (72mmol) of HOBt and 33.8 g (600mmol) of HBTU are weighed, dissolved by DMF, 15.6ml of DIPEA (72mmol) is added in an ice-water bath at 0 ℃, activated for 5 minutes and added into a reaction column, after 2 hours of reaction, 100ml of methanol and 100ml of DIPEA are added, mixed and sealed for 0.5 hour, DCM is washed for three times, the Resin is drained after methanol contraction, and 131g of Fmoc-Leu-2-Chlorotrityl Chloride Resin with the detection substitution degree of 0.46mmol/g is obtained.
Example 20 Boc-Phe-Phe-Gly-Ala-Ser (tBu) -Cys (Trt) -Leu-2-Chlorotrityl
Preparation of Chloride Resin
Weighing 109 g (50mmol) of Fmoc-Leu-2-Chlorotrityl Chloride Resin with the substitution degree of 0.48mmol/g in example 19 into a solid phase reaction column, adding DMF, and bubbling nitrogen for swelling for 60 minutes; the Fmoc protecting group was removed with DBLK and washed 6 times with DMF. Weighing 353 g (100mmol) of Fmoc-Leu-OH, 16.3g (120mmol) of HOBt and 38 g (100mmol) of HBTU, dissolving the mixture in DMF, adding 16.0 g of DIPEA (120mmol) at 0 ℃ in an ice-water bath, activating the mixture for 5 minutes, adding the mixture into a reaction column, reacting the mixture for 2 hours, washing the resin with DMF for three times, removing the Fmoc protecting group with DBLK, washing the resin with DMF for 6 times, and washing the resin with DCM for 3 times. The above coupling procedure was repeated, coupling Fmoc-Cys (Trt) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Boc-Phe-OH sequentially according to the peptide order. After completion of the reaction, the reaction mixture was shrunk with methanol and dried in vacuo to obtain 178g of a peptide resin.
Example 21 preparation of Boc-Phe-Phe-Gly-Ala-Ser (tBu) -Cys (Trt) -Leu-OH
178g of the peptide resin obtained in example 20 was placed in a 2000ml single neck flask, 1800ml of DCM solution prepared with 0.1% TFA was added to the flask, reacted at room temperature for 2.0 hours, the resin was filtered off, and the solvent was rotary evaporated to give 61g of white fully protected crude peptide.
2 2 2Example 22 preparation of Boc-Phe-Phe-Gly-Ala-Ser (tBu) -Cys (Trt) -Leu-NH- (CH) -NH
In a 500ml single-neck flask, 6.0g (100mmol) of ethylenediamine was added, dissolved in 50ml of DMF, and HOBt 16.3g (120mmol), and 15.1 g of DIC (120mmol) were added. 61g of the fully protected crude peptide from example 21 were dissolved in 200ml of DCM and added dropwise slowly to the ethylenediamine solution, after the dropwise addition was complete, the reaction was continued for 2h to terminate the reaction. 100ml of water was added to the reaction solution, the organic phase (DCM) was collected by extraction and rotary evaporation gave 42g of the crude white fully protected peptide of interest.
2 2Example 23 Boc-Phe-Phe-Gly-Ala-Ser (tBu) -Cys (Trt) -Leu-NH- (CH) -NH-L-
Preparation of Carnitine
In a 500ml single neck flask, 16.1g (100mmol) of L-Carnitine was charged, dissolved in 50ml of DMF, and 16.3g (120mmol) of HOBt and 15.1 g of DIC (120mmol) were added. 42g of the fully protected crude peptide obtained in example 22 was dissolved in 200ml of DCM, slowly added dropwise to the L-Carnitine solution, and after the dropwise addition was completed, the reaction was continued for 2h, and the reaction was completed. 100ml of water was added to the reaction mixture, and the organic phase (DCM) was collected by extraction and rotary evaporated to give Boc-Phe-Phe-Gly-Ala-Ser (tBu) -Cys (Trt) -Leu-NH- (CH)2)228g of-NH-L-Carnitine crude product.
2 2Example 24 preparation of crude H-Phe-Phe-Gly-Ala-Ser-Cys-Leu-NH- (CH) -NH-L-Carnitine
Preparation of
To the crude product from example 23 was added the pre-prepared lysate TFA: and (3) TIS: EDT (electro-thermal transfer coating): h2O85: 5:5:5 (vol.%) 50ml was reacted at room temperature for 2.0 hours, added to 5000ml of chilled dry ether to precipitate a white solid, centrifuged, the solid washed with dry ether and dried under vacuum to give 16.3g of a white solid, 35.13% yield and 64.5% HPLC purity.
2 2Example 25 purification of H-Phe-Phe-Gly-Ala-Ser-Cys-Leu-NH- (CH) -NH-L-Carnitine
16.3g of the crude product obtained in example 24 are taken, by means of a Waters 2454RP-HPLC system, wavelength 220nm, column chromatography 100X 500mm reversed phase C18 column, mobile phase: phase A: 0.3% TFA/acetonitrile (v/v); phase B: acetonitrile, gradient: b%: 38% -68%, flow rate: and collecting target peak fractions at 6 ml/min, performing rotary evaporation concentration, and freeze-drying to obtain 9.7g of target refined peptide, wherein the HPLC purity is 99.26%, and the total yield is 20.9%.
H-Phe-Phe-Gly-Ala-Ser-Cys-Leu-NH- (CH) was prepared by the same charge by the methods of examples 12-18 (invention) and examples 19-24 (Prior Art)2)2-NH-L-Carnitine, whose overall yield differs by a factor of approximately 3, by the process of the invention, although only the sequence of synthesis is changed, its overall yield far exceeds the expected yield.
2 2 6Example 26 preparation of NH- (CH) -NH-2-Chlorotrityl Chloride Resin
Weighing 200 g of 2-Chlorotrityl Chloride Resin with the substitution degree of 1.0mmol/g into a solid phase reaction column, adding DMF, and carrying out bubbling swelling for 60 minutes by nitrogen; weighing 46.4 g (400mmol) of hexamethylenediamine, dissolving with DMF, adding 90.0ml of DIPEA (500mmol) at 0 ℃ in an ice-water bath, adding into a reaction column, reacting for 1 hour, adding 200ml of methanol and 200ml of DIPEA, mixing and sealing for 0.5h, washing with DCM for three times, draining the resin after methanol contraction to obtain NH2-(CH2)6-NH-2-Chlorotrityl Chloride Resin。
2 6Example 27 preparation of Fmoc-Ser (tBu) -NH- (CH) -NH-2-Chlorotrityl Chloride Resin
Prepare for
NH obtained in example 26 was weighed2-(CH2)6Adding 100 grams of-NH-2-Chlorotrityl Chloride Resin into a solid phase reaction column, adding DMF, and carrying out bubbling with nitrogen for swelling for 60 minutes; Fmoc-Ser (tBu) -OH 22.8 g (60mmol), HOBt 9.72 g (72mmol) were weighed, dissolved in DMF and added to 16.4ml DIC (7 mmol) in an ice-water bath at 0 deg.C2mmol), activating for 5 minutes, adding into a reaction column, reacting for 2 hours, adding 70ml of acetic anhydride and 60ml of pyridine, mixing and sealing for 24 hours, washing with DCM for three times, contracting with methanol, and draining off the resin to obtain Fmoc-Ser (tBu) -NH- (CH)2)6-NH-2-Chlorotrityl Chloride Resin, detecting 116g of Resin with a degree of substitution of 0.55 mmol/g.
Example 28 Boc-Gly-His (Trt) -Trp (Boc) -Asp (OtBu) -Phe-Arg (Pbf) -Gln (Trt) -Arg
2 6Trp(Boc)-Trp(Boc)-Gln(Trt)-Pro-Ser(tBu)-Gly-Gly-Gly-Ser(tBu)-NH-(CH)-NH-2-
Preparation of Chlorotrityl Chloride Resin
The structure for preparing the target compound is as follows: GHWDFRQWWQPSGGGS-hexanediamine-Biotin.
Fmoc-Ser (tBu) -NH- (CH) with substitution degree of 0.55mmol/g in example 27 was weighed2)6Putting 91 g (50mmol) of-NH-2-Chlorotrityl Chloride Resin in a solid phase reaction column, adding DMF, and bubbling nitrogen for swelling for 60 minutes; the Fmoc protecting group was removed with DBLK and washed 6 times with DMF. Fmoc-Gly-OH 298 g (100mmol), HOBt 16.3g (120mmol), HBTU 38 g (100mmol) were weighed, dissolved in DMF, 16.0 g DIPEA (120mmol) was added in an ice water bath at 0 ℃ to activate for 5 min, the reaction column was added, after 2h reaction, the resin was washed three times with DMF, the Fmoc protecting group was removed with DBLK, washed 6 times with DMF and washed 3 times with DCM. The above coupling procedure was repeated, coupling Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH, Fmoc-Pro-OH, Fmoc-Gln (Trt) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Phe-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Trp (Boc) -OH, Fmoc-His (Trt) -OH, Boc-Gly-OH in the order of peptide sequence. After completion of the reaction, the reaction mixture was shrunk with methanol and dried in vacuo to obtain 274g of a peptide resin.
Example 29 Boc-Gly-His (Trt) -Trp (Boc) -Asp (OtBu) -Phe-Arg (Pbf) -Gln (Trt) -Arg
2 6 2Preparation of Trp (Boc) -Gln (Trt) -Pro-Ser (tBu) -Gly-Gly-Gly-Ser (tBu) -NH- (CH) -NH
Prepare for
274g of the peptide resin obtained in example 28 was placed in a 3000ml single-neck flask, 2800ml of a DCM solution prepared with 0.1% TFA was placed in the flask, and reacted at room temperature for 2.0 hours, after which the resin was filtered off and the solvent was evaporated off by rotary evaporation to obtain 169g of white fully protected crude peptide.
Example 30Boc-Gly-His (Trt) -Trp (Boc) -Asp (OtBu) -Phe-Arg (Pbf) -Gln (Trt) -Trp
2 6(Boc)-Trp(Boc)-Gln(Trt)-Pro-Ser(tBu)-Gly-Gly-Gly-Ser(tBu)-NH-(CH)-NH-Biotin
Preparation of
In a 1000ml single-neck flask were added 24.4g (100mmol) of Biotin, dissolved in 100ml of DMF, and 16.3g (120mmol) of HOBt and 15.1 g of DIC (120mmol) were added. The 169g of the fully protected crude peptide from example 29 was dissolved in 500ml of DCM, slowly added dropwise to the Biotin solution, and after the addition was completed, the reaction was continued for 2 hours to complete the reaction. Adding 100ml water into the reaction solution, extracting and collecting the organic phase (DCM), and rotary evaporating to obtain white Boc-Gly-His (Trt) -Trp (Boc) -Asp (OtBu) -Phe-Arg (Pbf) -Gln (Trt) -Trp (Boc) -Gln (Trt) -Pro-Ser (tBu) -Gly-Gly-Ser (tBu) -NH- (CH)2)6161g of-NH-Biotin crude product.
Example 31H-Gly-His-Trp-Asp-Phe-Arg-Gln-Trp-Trp-Gln-Pro-Ser-Gly-
6Preparation of Gly-Ser-NH- (CH2) -NH-Biotin crude product
To the crude product from example 30 was added the pre-prepared lysate TFA: and (3) TIS: EDT (electro-thermal transfer coating): h2O85: 5:5:5 (vol.%) 400ml, reacting at room temperature for 2.0 h, adding into 10000ml of frozen anhydrous ether, precipitating white solid, centrifuging, washing the solid with anhydrous ether, vacuum drying to obtain 92.6g of white solid, yield 83.7%, purity 56.4% by HPLC.
Example 32H-Gly-His-Trp-Asp-Phe-Arg-Gln-Trp-Trp-Gln-Pro-Ser-Gly-
6Purification of Gly-Ser-NH- (CH2) -NH-Biotin
92.6g of the crude product obtained in example 31 are taken, by means of a Waters 2454RP-HPLC system, wavelength 220nm, column chromatography 100X 500mm reversed phase C18 column, mobile phase: phase A: 0.3% TFA/acetonitrile (v/v); phase B: acetonitrile, gradient: b%: 38% -68%, flow rate: and collecting target peak fractions at 6 ml/min, performing rotary evaporation concentration, and freeze-drying to obtain 40.5g of target refined peptide, wherein the HPLC purity is 98.90%, and the total yield is 36.6%.
Example 33 preparation of Fmoc-Ser (tBu) -2-Chlorotrityl Chloride Resin
Weighing 100 g of 2-Chlorotrityl Chloride Resin with the substitution degree of 1.0mmol/g into a solid phase reaction column, adding DMF, and carrying out bubbling swelling for 60 minutes by nitrogen; Fmoc-Ser (tBu) -OH 22.8 g (60mmol), HOBt 9.72 g (72mmol) and HBTU 33.8 g (600mmol) are weighed, dissolved in DMF, 15.6ml DIPEA (72mmol) is added in ice water bath at 0 ℃, activated for 5 minutes, added into a reaction column, reacted for 2 hours, 100ml methanol and 100ml DIPEA are added, mixed and sealed for 0.5 hour, DCM is washed for three times, the Resin is drained after methanol contraction to obtain Fmoc-Leu-2-Chlorotrityl Chloride Resin, and 139g of the Resin with the detection substitution degree of 0.45mmol/g is detected.
Example 34 Boc-Gly-His (Trt) -Trp (Boc) -Asp (OtBu) -Phe-Arg (Pbf) -Gln (Trt) -Arg
Trp(Boc)-Trp(Boc)-Gln(Trt)-Pro-Ser(tBu)-Gly-Gly-Gly-Ser(tBu)-2-Chlorotrityl
Preparation of Chloride Resin
111 g (50mmol) of Fmoc-Ser (tBu) -2-Chlorotrityl Chloride Resin with the substitution degree of 0.45mmol/g in example 33 is weighed into a solid phase reaction column, DMF is added, and nitrogen is bubbled for swelling for 60 minutes; the Fmoc protecting group was removed with DBLK and washed 6 times with DMF. Fmoc-Gly-OH 298 g (100mmol), HOBt 16.3g (120mmol), HBTU 38 g (100mmol) were weighed, dissolved in DMF, 16.0 g DIPEA (120mmol) was added in an ice water bath at 0 ℃ to activate for 5 min, the reaction column was added, after 2h reaction, the resin was washed three times with DMF, the Fmoc protecting group was removed with DBLK, washed 6 times with DMF and washed 3 times with DCM. The above coupling procedure was repeated, coupling Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ser (tBu) -OH, Fmoc-Pro-OH, Fmoc-Gln (Trt) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Phe-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Trp (Boc) -OH, Fmoc-His (Trt) -OH, Boc-Gly-OH in the order of peptide sequence. After completion of the reaction, the reaction mixture was shrunk with methanol and dried in vacuo to obtain 268g of a peptide resin.
Example 35 Boc-Gly-His (Trt) -Trp (Boc) -Asp (OtBu) -Phe-Arg (Pbf) -Gln (Trt) -Arg
Preparation of Trp (Boc) -Gln (Trt) -Pro-Ser (tBu) -Gly-Gly-Gly-Ser (tBu) -OH
268g of the peptide resin from example 34 was charged into a 3000ml single neck flask, 2800ml of a solution of 0.1% TFA in DCM was charged into the flask, reacted at room temperature for 2.0 hours, the resin was filtered off and the solvent was rotary evaporated to give 158g of white fully protected crude peptide.
Example 36 Boc-Gly-His (Trt) -Trp (Boc) -Asp (OtBu) -Phe-Arg (Pbf) -Gln (Trt) -Arg
2 6 2Preparation of Trp (Boc) -Gln (Trt) -Pro-Ser (tBu) -Gly-Gly-Gly-Ser (tBu) -NH- (CH) -NH
Prepare for
In a 500ml single-neck flask, 11.6.0g (100mmol) of hexamethylenediamine was charged, dissolved in 50ml of DMF, and 16.3g (120mmol) of HOBt and 15.1 g of DIC (120mmol) were added. 61g of the fully protected crude peptide from example 21 were dissolved in 200ml of DCM and added dropwise slowly to the hexamethylenediamine solution, and after completion of the addition, the reaction was continued for 2 hours to terminate. 100ml of water was added to the reaction solution, the organic phase (DCM) was collected by extraction and rotary evaporation gave 158g of the crude white fully protected peptide of interest.
Example 37 Boc-Gly-His (Trt) -Trp (Boc) -Asp (OtBu) -Phe-Arg (Pbf) -Gln (Trt) -Arg
2 6Trp(Boc)-Trp(Boc)-Gln(Trt)-Pro-Ser(tBu)-Gly-Gly-Gly-Ser(tBu)-NH-(CH)-NH-
Preparation of Biotin
In a 1000ml single-neck flask were added 24.4g (100mmol) of Biotin, dissolved in 100ml of DMF, and 16.3g (120mmol) of HOBt and 15.1 g of DIC (120mmol) were added. 158g of the fully protected crude peptide obtained in example 36 were dissolved in 500ml of DCM and slowly added dropwise to the Biotin solution, and after the addition was completed, the reaction was continued for 2 hours to complete the reaction. Adding 100ml water into the reaction solution, extracting and collecting the organic phase (DCM), and rotary evaporating to obtain white Boc-Gly-His (Trt) -Trp (Boc) -Asp (OtBu) -Phe-Arg (Pbf) -Gln (Trt) -Trp (Boc) -Gln (Trt) -Pro-Ser (tBu) -Gly-Gly-Ser (tBu) -NH- (CH)2)6126g of-NH-Biotin crude product.
Example 38H-Gly-His-Trp-Asp-Phe-Arg-Gln-Trp-Trp-Gln-Pro-Ser-Gly-
6Preparation of Gly-Ser-NH- (CH2) -NH-Biotin crude product
To the crude product from example 37 was added the pre-prepared lysate TFA: and (3) TIS: EDT (electro-thermal transfer coating): h2O85: 5:5:5 (volume ratio) 400ml, reacting at room temperature for 2.0 hours, adding into 10000ml of frozen anhydrous ether, precipitating white solid, centrifuging, washing the solid with anhydrous ether, and drying in vacuum to obtain 45.9g of white solid, the yield is 41.5%, and the HPLC purity is 50.7%.
Example 39H-Gly-His-Trp-Asp-Phe-Arg-Gln-Trp-Trp-Gln-Pro-Ser-Gly-
6Purification of Gly-Ser-NH- (CH2) -NH-Biotin
45.9g of the crude product obtained in example 38 were taken, using a Waters 2454RP-HPLC system, wavelength 220nm, column 100X 500mm reversed phase C18 column, mobile phase: phase A: 0.3% TFA/acetonitrile (v/v); phase B: acetonitrile, gradient: b%: 38% -68%, flow rate: and collecting target peak fractions at 6 ml/min, performing rotary evaporation concentration, and freeze-drying to obtain 16.3g of target refined peptide, wherein the HPLC purity is 99.2%, and the total yield is 14.7%.
GHWDFRQWWQPSGGGS-hexanediamine-Biotin was prepared in a total yield of 2 times more by the same charge by the methods of examples 26-33 (inventive) and examples 34-39 (prior art), and by the method of the present invention, although only the order of synthesis was changed, the total yield far exceeded the expected yield.
Claims (17)
1. A method for synthesizing C-terminal modified peptide, which is characterized by comprising the following steps:
1) coupling one end amino group of the diamino compound to the solid-phase synthetic resin;
2) adopting Fmoc solid-phase peptide synthesis strategy to couple amino acid to the amino group at the other end of the diamino compound in sequence to obtain fully-protected polypeptide resin;
3) cleaving the fully protected polypeptide from the resin to obtain a fully protected polypeptide;
4) deprotecting the fully protected polypeptide to obtain the desired C-terminally modified peptide, or
Coupling the fully protected polypeptide with a carboxyl-containing modification group to obtain the target C-terminal modified peptide.
2. The method for synthesizing a C-terminally modified peptide according to claim 1, wherein the diamino compound is selected from the group consisting of linear symmetrical diamino compounds or branched symmetrical diamino compounds or sterically symmetrical diamino compounds.
3. The method for synthesizing a C-terminally modified peptide according to claim 2, wherein the diamino compound is selected from the group consisting of ethylenediamine, propylenediamine, butylenediamine, pentylenediamine, hexylenediamine, heptylenediamine, octylenediamine, and mixtures thereof,
4. The method for synthesizing a C-terminal modified peptide according to claim 1, wherein the solid phase synthetic Resin is selected from 2-Chlorotrityl Chloride Resin.
5. The method for synthesizing C-terminal modified peptide according to claim 4, wherein the degree of substitution of the solid phase synthetic resin is in the range of 0.1 to 1.2 mmol/g.
6. The method for synthesizing C-terminal modified peptide according to claim 5, wherein the degree of substitution of the solid phase synthetic resin is in the range of 0.2 to 0.8 mmol/g.
7. The method for synthesizing C-terminal modified peptide according to claim 6, wherein the degree of substitution of the solid phase synthetic resin is in the range of 0.3 to 0.5 mmol/g.
8. The method for synthesizing C-terminally modified peptides according to claim 1, wherein the coupling system used in the Fmoc solid phase peptide synthesis strategy of step 2) is DIC + A or B + A + C, wherein A is HOBt or HOAt, B is HBTU, HATU, TBTU or PyBOP, and C is DIPEA or TMP.
9. The method for synthesizing C-terminal modified peptide according to claim 1, wherein the cleavage reagent used in the cleavage step of step 3) is TFA, TIS, EDT, H2The cracking time of the O composition is 1.5-3.5 hours.
10. The method for synthesizing a C-terminal modified peptide according to claim 9, wherein the volume ratio of the cleavage reagent is TFA: and (3) TIS: EDT (electro-thermal transfer coating): h2O＝85-95:2-5:2-5:1-5。
11. The method for synthesizing a C-terminally modified peptide according to claim 1, further comprising a purification step after step 4).
12. The method for synthesizing a C-terminal modified peptide according to claim 11, wherein the purification step is purification by HPLC.
13. The method for synthesizing C-terminal modified peptide according to any one of claims 1 to 12, wherein the step 1) is performed by weighing the solid-phase synthetic resin, placing the solid-phase synthetic resin in a solid-phase reaction column, adding DMF, and carrying out bubbling and swelling with nitrogen; weighing a diamino compound, dissolving the diamino compound with DMF (dimethyl formamide), adding DIPEA into a solid-phase reaction column, adding methanol and DIPEA after complete reaction, mixing and sealing for 10-60 minutes, washing with DCM (diethyl formamide), and draining the resin after methanol shrinkage to obtain the diamino compound coupled solid-phase synthetic resin.
14. The method for synthesizing a C-terminally modified peptide according to any of claims 1-12, wherein the step of coupling the first amino acid to the other amino group of the diamino compound in step 2) is: adding DMF into the diamino compound coupled solid-phase synthetic resin obtained in the step 1), and carrying out nitrogen bubbling for swelling; activating the amino acid protected by Fmoc by using a coupling system, adding the activated amino acid protected by Fmoc into a reaction column for reaction, then adding acetic anhydride and pyridine, mixing and sealing to obtain the amino acid diamino compound coupled solid-phase synthetic resin protected by Fmoc.
15. The method for synthesizing a C-terminal modified peptide according to any one of claims 1 to 12, wherein the amino acid sequence on the amino group sequentially coupled to the other end of the amino compound is selected from polypeptides comprising 1 to 20 amino acids.
16. The method for synthesizing a C-terminal modified peptide according to claim 15, wherein the amino acid sequence coupled to the other end of the amino compound in sequence is selected from polypeptides comprising 5 to 15 amino acids.
17. The method for synthesizing C-terminal modified peptide according to any one of claims 1 to 12, wherein the carboxyl-containing modifying group is selected from Biotin, fluorescein, carnitine, formic acid, acetic acid, palmitic acid, stearic acid, cholic acid, carboxyl-containing small molecule drugs and pharmacodynamic functional groups.
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| CN112940103A (en) * | 2019-12-10 | 2021-06-11 | 深圳翰宇药业股份有限公司 | Synthetic method of teriparatide impurity F |
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| CN114685616B (en) * | 2020-12-31 | 2024-03-29 | 哈尔滨三联药业股份有限公司 | Synthesis method of triptorelin acetate |
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