CN109988062B - Liquid phase spherical carrier and preparation method and application thereof - Google Patents
Liquid phase spherical carrier and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a liquid phase spherical carrier and a preparation method and application thereof, and particularly discloses a spherical carrier for liquid phase synthesis, which has a result formula shown as a formula I:wherein R is a functional group, preferably selected from a group having a hydroxyl group, an amino group, a carboxyl group or a halogen. The liquid phase synthesis carrier can be used for liquid phase synthesis of polypeptide, and has simple preparation method and high synthesis efficiency.
Description
Technical Field
The invention relates to the field of polypeptide synthesis, in particular to a liquid phase spherical carrier and a preparation method and application thereof.
Background
Polypeptide synthesis has been known for over 100 years, and at the present stage, the traditional solid-phase synthesis method and liquid-phase synthesis method are mainly adopted. Both of these methods have different disadvantages. The traditional liquid phase synthesis method has the defects of multiple reaction steps, difficult separation method, complex post-treatment, long synthesis period and the like; after each peptide grafting, the separation, purification or crystallization is carried out to remove unreacted raw materials or reaction byproducts, which is time-consuming and troublesome, and the loss caused by the operation is large; to overcome these disadvantages, Merrifield first developed a method for solid phase polypeptide synthesis in 1963. The solid phase synthesis method can avoid complicated steps of separation and purification, and has obvious superiority compared with a liquid phase method: (1) in the process of polypeptide synthesis, a peptide chain is connected to an insoluble carrier, so that a product peptide is also insoluble and is easy to wash and filter; (2) the reaction can be completed by adding excessive reactants, and excessive reagents and byproducts can be removed by washing and filtering; (3) the whole reaction is carried out in the same container, so that the mechanical loss caused by multiple times of precipitation, washing and separation in the liquid phase synthesis is avoided, and the operation is simple, convenient and quick; (4) the operation has strong repeatability, and is beneficial to the automation of polypeptide synthesis reaction. At the present stage, the polypeptide synthesis mostly adopts a solid-phase synthesis method. However, solid phase synthesis requires the use of solid phase support resins, and the main solid phase support resins at present are: polystyrene-divinylbenzene crosslinked resins; a polyamide resin; polyethylene-ethylene glycol resins, and the like. The polypeptide synthesized by the solid phase method cannot effectively monitor the intermediate reaction step and effectively separate impurities generated in the process, so that the impurity components in the final product are complex and the separation and purification difficulty is high; because a solid phase carrier is used, the synthesis scale is influenced by factors such as resin substitution degree and the like, resin granularity, mechanical properties and the like, and is generally obviously smaller than that of a liquid phase method.
Structural unit of polystyrene and divinylbenzene crosslinked resin
For the conventional liquid phase synthesis method, the synthesis of long peptides is often difficult, the workload is large, the separation and purification work is difficult, and other methods are generally selected for polypeptide synthesis in the synthesis of long peptides. The traditional solid phase synthesis method generally adopts a polymer as a carrier, introduces related functional groups on the carrier, and then carries out a series of derivatization to synthesize polypeptides with different groups, but for the solid phase method, the carrier is a polymer, so that the structure of the carrier is difficult to indicate, and the functional groups are not uniformly distributed on the surface of resin, so that the defects exist. These defects lead to coupling difficulties and impurities, which are particularly difficult for long peptide synthesis, and the development of new supports becomes an important task for solid phase synthesis. In order to improve the shortcomings of the conventional solid phase synthesis methods in recent years, scientists invented a class of polypeptide compound synthesis using a combination of liquid phase synthesis and solid phase support. The polypeptide is synthesized by designing a liquid phase carrier with a specific structure, performing amino acid coupling in a liquid phase, and combining a solid phase synthesis and liquid phase synthesis intermediate purification and post-treatment method. Such methods can greatly improve the scale of production of solid phase synthesis and the purity of intermediates. Realizes the amplification of the polypeptide synthesis scale and greatly improves the production efficiency. However, it is difficult to find a suitable solid support with small steric hindrance, and it is critical to find a good support compound by either solid support synthesis or liquid support synthesis.
The solid phase carrier has uneven surface distribution, the solid phase resin carrier particles have different sizes, the resins purchased in the market are all products with a particle size range, the resins often cause mechanical damage, cracks, collapse and the like in the production process, and the reaction can not be effectively monitored in the application process; the liquid phase carrier is generally a planar molecule, and as the steric hindrance between peptide chain residues is increased along with the growth of a peptide chain, side reactions are increased, which affects the synthesis efficiency. According to experiments, the chloro fullerene has good performance, on one hand, fullerene molecules are in a regular spherical structure, functional groups are distributed on the surface of a sphere, the position of each functional group can be monitored, and on the other hand, the sphere shows that the intermolecular resistance of the functional groups is slowly increased along with the increase of peptide chains in the coupling process, so that the utilization rate of molecular space is maximized.
C60Cl6Molecular structural formula (II)
The surface substituent of the solid phase synthesis carrier can not be effectively monitored, the particle size distribution of the carrier is not uniform, the surface of the carrier collapses, the reaction efficiency is influenced, and the surface resistance of the polypeptide on the solid phase carrier increases along with the growth of the peptide chain. Common solid support functional groups are as follows:
disclosure of Invention
In order to solve the above problems, an aspect of the present invention provides a spherical support for liquid phase synthesis, having a resultant formula shown in formula I:
wherein R is a functional group.
In the technical scheme of the invention, the R group is selected from a group with hydroxyl, amino, carboxyl or halogen.
n is any integer from 1 to 4.
In another aspect, the present invention provides a method for preparing a spherical support for liquid phase synthesis, which comprises the steps of:
get C60Cl6And p-hydroxymethyl phenol or p-hydroxyethyl phenol or p-hydroxypropyl phenol or p-hydroxybutyl phenol in organic solvent and potassium carbonateOr
C is to be60Cl6And compoundsPerforming substitution reaction under the condition of potassium carbonate, after the reaction is completed, hydrogenating in sodium borohydride and ethanol until the reaction is completed, adding methanesulfonic acid and Fmoc-NH2Reacting under the condition of sodium carbonate to be complete, and removing Fmoc protecting group to obtain
C is to be60Cl6With Fmoc-NH2Carrying out substitution reaction under the condition of potassium carbonate; then removing Fmoc protecting group, and reacting withCarrying out condensation reaction under the action of a condensing agent under the alkaline condition, and removing Fmoc protecting group to obtain
In another aspect, the invention provides the use of a compound of formula I as a support for liquid phase synthesis.
In a further aspect, the present invention provides the use of a compound of formula I for the synthesis of polypeptide chains in the liquid phase.
In another aspect, the invention provides a method for synthesizing a polypeptide, which uses a compound shown in formula I as a liquid phase synthesis carrier, and sequentially couples amino acids on functional groups of the compound shown in formula I.
In the technical scheme of the invention, the method for coupling the amino acid comprises the steps of activating a carboxyl component by using a condensing agent, condensing the carboxyl component and an amino component under an alkaline condition, removing an amino protecting group, continuously coupling the next amino acid until the polypeptide is finished, and synthesizing a carrier by a cracking liquid phase.
In the technical scheme of the invention, the condensing agent is selected from one or more of EDCI, EDC, DCC, DIC, HATU, HBTU, HOAt, HOBt, PyAOP and PyBOP.
In the technical scheme of the invention, the alkaline condition is provided by one or more of DIEA, NMM, TEA, pyridine, DBU, N-methylmorpholine, collidine or lutidine.
In the technical scheme of the invention, the condition for removing the amino protecting group is alkaline, and the removal of the amino protecting group by diethylamine and DBU is preferred.
In the technical scheme of the invention, the amino protecting group is selected from Fmoc or Boc.
In the technical scheme of the invention, the conditions of the cleaved polypeptide and the liquid phase synthesis carrier are TFA, TIS and H2A combination of O, preferably TFA, TIS, H2O=95:2.5:2.5。
In the technical scheme of the invention, the compound shown in the formula I has one or more functional groups of amino, carboxyl, hydroxyl and halogen.
The invention further provides a method for synthesizing bivalirudin, which takes a compound shown as a formula I as a liquid phase synthesis carrier, couples Fmoc-Leu-OH, removes amino protecting groups, sequentially couples Fmoc-Tyr (tBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Phe-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg-OH, Pbf) -OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Ile, Boc-D-Phe-OH, removing protective group, synthesizing the carrier by lysate phase, and purifying to obtain bivalirudin.
Advantageous effects
1) The spherical molecules are uniform in size, and the substituent groups can be judged on the surface of the sphere;
2) the spherical liquid phase carrier has no structural defect, and the reaction is not difficult due to the structural defect in the reaction process;
3) the medium resistance of the reaction of the spherical molecule surface functional groups is small; is convenient for industrial production and application.
Drawings
FIG. 1 is a mass spectrum of Compound A.
FIG. 2 is the mass spectrum of compound B.
FIG. 3 is a mass spectrum of Compound C.
FIG. 4 is a mass spectrum of bivalirudin crude peptide.
FIG. 5 is a HPLC chart of purified bivalirudin.
Detailed Description
The invention is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only, and are not intended to limit the scope of the present invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
EXAMPLE 1 Synthesis of Compound A
Taking a compound 1 (C)60Cl6)5.6 g (6mmol) and 7.4g (60mmol) of p-hydroxymethylphenol were charged into a 500mL three-necked flask, and then DMF (100mL) was added to the reaction flask, followed by stirring and addition of 8.3 g (60mmol) of potassium carbonate. The reaction was warmed to 80 ℃ and stirred for 16 hours. The reaction was monitored by HPLC. After the raw material was consumed, the reaction solution was cooled to below 10 ℃ in an ice bath. Slowly dripping 1mol/L diluted solution into the reaction solution under the condition of fully stirringHydrochloric acid (100mL), and purified water (200mL), and stirring was continued for half an hour after the addition was complete. After the solid had precipitated, it was filtered, and the cake was washed with purified water (100mL) and ether (100mL) in this order. Drying at 60 ℃ for 5 hours under vacuum gave 7.0g of compound a in 90% yield. The molecular weight is 1371.586Da [ M + H ] confirmed by mass spectrum+]The compound A is the Wang resin type liquid phase carrier.
EXAMPLE 2 Synthesis of Compound B
14.7 g (5mmol) of the compound and 212.9 g (50mmol) of the compound were charged in a 1L three-necked flask, and then DMF (150mL) was added to the reaction flask, followed by stirring and addition of 6.9 g (50mmol) of potassium carbonate. The reaction was warmed to 80 ℃ and stirred for 16 hours. The reaction was monitored by HPLC. After the raw material was consumed, the reaction solution was cooled to below 10 ℃ in an ice bath. 1mol/L dilute hydrochloric acid (100mL) and purified water (150mL) were slowly added dropwise to the reaction mixture with sufficient stirring, and stirring was continued for half an hour after the addition was completed. Filtration and the filter cake was washed successively with purified water (100mL) and diethyl ether (100 mL). Vacuum drying at 60 deg.C for 5 hr gave 3 as a pale yellow solid, 8.8g, 90% yield.
38.8 g (4.5mmol) of the compound was weighed into a 1L three-necked flask, and then THF (100mL) and methanol (100mL) were added to the reaction flask and stirred uniformly. After the reaction solution was warmed to 60 ℃, sodium borohydride (3.1g, 50mmol) was slowly added, and after the addition was completed, the temperature was maintained and stirring was continued for 4 hours. The reaction was monitored by TLC (ethyl acetate: n-hexane ═ 1:2, uv lamp color development). After the raw material was consumed, the reaction solution was cooled to below 10 ℃ in an ice bath. Under sufficient stirring, 1mol/L dilute hydrochloric acid (100mL) was slowly added dropwise, after completion of the addition, THF was removed by concentration under reduced pressure, purified water (450mL) was added to the remaining reaction solution, and the pH was adjusted to 5-7 with 1mol/L dilute hydrochloric acid. Filtration and the filter cake was washed sequentially with purified water (100mL) and methanol (100 mL). Vacuum drying at 60 deg.C for 8 hr gave 4, 8.6g of pale yellow compound as a solid in 98% yield.
Compound 4(8.6g,4.4mmol) was weighed into a 1L three-necked flask, toluene (400mL) was added to the flask, the mixture was stirred well, and methanesulfonic acid (2.3 g,25mmol) and Fmoc-NH2(8.9 g, 40mmol) were added sequentially. The reaction solution was heated to 110 ℃ and stirred for 3 hours. The reaction was cooled to room temperature, sodium carbonate (2.6g,25mmol) was added, and the solvent was removed by rotary evaporation. Methanol (400mL) and toluene (100mL) were added to the residue, the temperature was raised to 90 ℃ and the solution was stirred. And naturally cooling the reaction solution to room temperature for crystallization. And (5) filtering. The filter cake was washed with a mixed solvent of methanol and acetonitrile (1:1, 100mL), and vacuum-dried at 50 ℃ for 5 hours to obtain 512.8 g of a yellow solid compound with a yield of 95%.
Weighing 512.8 g (4.2mmol) of the compound, adding the compound into a 1L three-neck flask, adding chloroform (100mL) into a reaction bottle, stirring to dissolve the compound clearly, adding DBU (6.0g, 40mmol), cooling the reaction liquid to below 5 ℃ in an ice bath, slowly adding diethylamine (7.4g,100mmol) dropwise, controlling the temperature to be not more than 5 ℃, after the dropwise addition is finished, raising the temperature of the reaction liquid to room temperature, continuing stirring for 2 hours, monitoring the reaction by TLC (DCM: MeOH: HAc ═ 100:1:0.5), after the reaction is finished, concentrating the reaction liquid at 30 ℃ under reduced pressure to obtain a viscous substance, adding acetonitrile (20mL) into the viscous substance, stirring for 30 minutes, filtering, washing the filter cake twice by methanol (10mL × 2), and drying the filter cake at 40 ℃ under vacuum for 2 hours to obtain 8.1g of a light yellow solid, the yield is 98%, and the molecular weight is confirmed by mass spectrometry to be 2046.642Da [ M + H (M+]. The yellow solid is the compound B. The compound B is an amino resin type liquid phase carrier.
EXAMPLE 3 Synthesis of Compound C
The compound (16.6 g, 7mmol) and Fmoc-NH were taken216.7 g (70mmol) was charged in a 1L three-necked flask, and then DMF (150mL) was added to the reaction flask, followed by stirring and addition of 6.9 g (50mmol) of potassium carbonate. The reaction was warmed to 80 ℃ and stirred for 16 hours. The reaction was monitored by HPLC. After the raw material was consumed, the reaction solution was cooled to below 10 ℃ in an ice bath. 1mol/L dilute hydrochloric acid (100mL) and purified water (150mL) were slowly added dropwise to the reaction mixture with sufficient stirring, and stirring was continued for half an hour after the addition was completed. Filtering, filteringThe cake was washed successively with purified water (100mL) and diethyl ether (100 mL). Vacuum drying at 60 ℃ for 5 hours gave compound 6 as a brown solid in 92% yield, 12.5 g.
612.5 g (6.5mmol) of the compound was weighed into a 1L three-necked flask, chloroform (150mL) was added to the flask, and then DBU (6.0g, 40mmol) was added thereto with stirring. The reaction was cooled to below 5 ℃ in an ice bath and diethylamine (11.1g,150mmol) was slowly added dropwise with the temperature controlled not to exceed 5 ℃. After the addition was complete, the reaction was allowed to warm to room temperature and stirring was continued for 2 hours. The reaction was monitored by TLC (DCM: MeOH: HAc: 100:1: 0.5). After completion of the reaction, the reaction mixture was concentrated under reduced pressure at 30 ℃ to give a viscous product, and acetonitrile (20mL) was added to the viscous product, followed by stirring for 30 minutes. Filtration and the filter cake was washed twice with methanol (10 mL. times.2). The filter cake was dried under vacuum at 40 ℃ for 2 hours to give 5.5g of a pale yellow solid with a yield of 98%. The yellow solid is compound 7.
Compound 7(5.5g,6.4mmol) was weighed into a 250mL three-necked flask, chloroform (100mL) was added to the flask, and HOBt (5.2g.38mmol) and bromoacetic acid (5.3g,38mmol) were sequentially added thereto. Stirring and dissolving to clear. EDC. HCl (7.3g,38mmol) was added and stirring continued at room temperature for 3 h. TLC monitored the reaction. After the reaction is completed, the reaction solution is decompressed and concentrated to be viscous at the temperature of 30 ℃, and the mixed solvent of chloroform and petroleum ether is recrystallized. The product is washed three times by a chloroform petroleum ether mixed solution. The filter cake was dried under vacuum at 40 ℃ for 5 hours to give 88.9 g of compound in 96% yield.
88.9 g (6.1mmol) of compound and 918.3 g (38mmol) of compound were charged in a 1L three-necked flask, and then DMF (150mL) was added to the reaction flask, followed by stirring and addition of 5.3g (38mmol) of potassium carbonate. The reaction was warmed to 80 ℃ and stirred for 16 hours. The reaction was monitored by HPLC. After the raw material was consumed, the reaction solution was cooled to below 10 ℃ in an ice bath. 1mol/L dilute hydrochloric acid (100mL) and purified water (150mL) were slowly added dropwise to the reaction mixture with sufficient stirring, and stirring was continued for half an hour after the addition was completed. Filtration and the filter cake was washed successively with purified water (100mL) and diethyl ether (100 mL). Vacuum drying at 60 ℃ for 5 hours gave compound 10 as a yellow solid, 20.3g, 96% yield.
Weighing 1020.3 g (5.8mmol) of compound, adding the compound into a 1L three-neck flask, adding chloroform (150mL) into a reaction bottle, stirring to dissolve the mixture, adding DBU (6.0g, 40mmol), cooling the reaction liquid to below 5 ℃ in an ice bath, slowly adding diethylamine (7.4g,100mmol) dropwise, controlling the temperature to be not more than 5 ℃, after the dropwise addition is finished, heating the reaction liquid to room temperature, continuing stirring for 2 hours, monitoring the reaction by TLC, concentrating the reaction liquid at 30 ℃ under reduced pressure to form a viscous substance, adding acetonitrile (20mL) into the viscous substance, stirring for 30 minutes, filtering, washing the filter cake twice by methanol (10mL × 2), drying the filter cake at 40 ℃ for 2 hours in vacuum to obtain 13.4g of light yellow solid, wherein the yield is 98%, and the yellow solid is the compound which has the molecular weight of 2331.476Da [ M + H ] confirmed by mass spectrum+]. The compound C is an amino resin type liquid phase carrier.
Example 4 vectors for polypeptide Synthesis
The Wang resin type carrier is used for synthesizing a specific peptide sequence D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-u-Tyr-Leu-OH (bivalirudin).
Coupling the first amino acid (Fmoc-Leu-OH)
Compound A (7.0g,5.4mmol) was charged into a 250mL three-necked flask, chloroform (200mL) was added to the flask, and HOBt (4.05g.30mmol) and DMAP (0.8 g, 6mmol) were sequentially added Fmoc-Leu-OH (10.7g,30 mmol). Stirring and dissolving to clear. EDC. HCl (5.8g,30mmol) was added and stirring continued at room temperature for 3 h. The reaction was monitored by HPLC. After completion of the reaction, the reaction mixture was concentrated under reduced pressure at 30 ℃ to give a viscous product, and methanol (60mL) was added to the viscous product, followed by stirring for 2 hours. Filtration and washing of the filter cake three times with methanol (20 mL. times.3). And drying the filter cake at 40 ℃ for 5 hours in vacuum to obtain a compound D.
Coupling of the second amino acid (Fmoc-Tyr (tBu) -OH)
Compound D (5.4mmol) was placed in a 100mL three-necked flask, chloroform (100mL) was added to the flask, and then DBU (4.8g, 30mmol) was added thereto with stirring. The reaction was cooled to below 5 ℃ in an ice bath and diethylamine (4.1g,54mmol) was slowly added dropwise with the temperature controlled not to exceed 5 ℃. After the addition was complete, the reaction was allowed to warm to room temperature and stirring was continued for 2 hours. The reaction was monitored by HPLC. After completion of the reaction, the reaction mixture was concentrated under reduced pressure at 30 ℃ to give a viscous product, and acetonitrile (20mL) was added to the viscous product, followed by stirring for 30 minutes. Filtration and the filter cake was washed twice with methanol (10 mL. times.2). The filter cake was dried under vacuum at 40 ℃ for 2 hours to give off-white solid E.
Solid compound E was charged into a 1000mL three-necked flask, chloroform (200mL) was added to the reaction flask, and HOBt (4.05g.30mmol) and Fmoc-Tyr (tBu) -OH (13.8g,30mmol) were sequentially added thereto. Stirring and dissolving to clear. The reaction was cooled to 0 ℃. EDC. HCl (5.8g,30mmol) was added and stirring was continued at 0-10 ℃ for 3 hours. The reaction was monitored by HPLC. After completion of the reaction, the reaction mixture was concentrated under reduced pressure at 30 ℃ to give a viscous product, and methanol (20mL) was added to the viscous product, followed by stirring for 2 hours. Filtration and washing of the filter cake three times with methanol (10 mL. times.3). And drying the filter cake for 3 hours under vacuum at 40 ℃ to obtain the compound F.
The coupling reaction conditions of Fmoc-Tyr (tBu) -OH were repeated, and Fmoc-Glu (OtBu) -OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Phe-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Gly-OH, Fmoc-Asn (Trt) -OH, Fmoc-Gly-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH and Boc-D-Phe-OH were coupled in this order according to the peptide sequence to obtain 58.6g of peptide-linked carrier compound P, with a synthesis yield of 79%.
The above-mentioned white solid compound P (58.6g) was added to the lysate (TFA: EDT: H)2O90: 5:5, 300mL), stirring at room temperature for 2 hours, then slowly pouring the reaction solution into frozen ethyl ether (5L), stirring for 30 minutes, standing in a refrigerator for 1 hour, centrifuging, washing with ethyl ether (50mL × 3) for three times, drying the obtained precipitate at 30 ℃ for 2 hours, pulping with methanol (10mL) for 2 hours, filtering, removing the filter cake, and spin-drying the filtrate at 40 ℃ to obtain 36.4g of white solid, wherein the yield of the crude peptide is 75%, and the mass spectrum molecular weight of the crude peptide is 2180.350Da and is consistent with the target molecular weight.
36.4g of the crude peptide are dissolved in 500ml of water. The obtained crude peptide solution is purified by a NOVASEP RP-HPLC system with the wavelength of 220nm and a reversed phase C18 chromatographic column, and is purified by a conventional 0.1% TFA/water and acetonitrile mobile phase system, salt is converted, target peak fractions are collected, concentrated by rotary evaporation and freeze-dried to obtain 30.2g of refined peptide, the HPLC purity is more than 99.5%, the maximum single impurity is less than 0.1%, and the total yield is 64.8%. See fig. 5.
Patent CN 102286076 a reports a solid phase synthesis method of bivalirudin, with a yield of 55.8%, 608 g of refined peptide is obtained by using wang resin 500 g, 1mmol/g substitution degree. The method only needs 7.0g of carrier to obtain 30.2g of propeptide, only needs 140 g of carrier to obtain 608 g of propeptide by amplification according to the quantity, and the carrier has higher molecular utilization rate. The carrier has obvious advantages from yield and carrier molecule utilization rate, and has wide industrial prospect.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (7)
2. The method for preparing a spherical carrier according to claim 1, comprising the steps of:
get C60Cl6And p-hydroxymethyl phenol or p-hydroxyethyl phenol or p-hydroxypropyl phenol or p-hydroxybutyl phenol in organic solvent and potassium carbonateOr
C is to be60Cl6And compoundsPerforming substitution reaction under the condition of potassium carbonate, after the reaction is completed, hydrogenating in sodium borohydride and ethanol until the reaction is completed, adding methanesulfonic acid and Fmoc-NH2Reacting under the condition of sodium carbonate to be complete, and removing Fmoc protecting group to obtainOr
C is to be60Cl6With Fmoc-NH2Carrying out substitution reaction under the condition of potassium carbonate; then removing Fmoc protecting group, and reacting withCarrying out condensation reaction under the action of a condensing agent under the alkaline condition, and removing Fmoc protecting group to obtain
3. Use of the spherical support according to claim 1 for the synthesis of polypeptide chains in liquid phase.
4. Use of the spherical support according to claim 1 as a support for liquid phase synthesis.
5. A method for synthesizing polypeptide, which comprises using the spherical carrier of claim 1 as a liquid phase synthesis carrier, and sequentially coupling amino acids to the functional groups of the compound represented by formula I.
6. The method of claim 5, wherein the amino acid is coupled by activating the carboxyl component with a condensing agent, condensing with the amino component under alkaline conditions to remove the amino protecting group, and continuing to couple the next amino acid until the polypeptide is completed, and lysing the liquid phase to synthesize the carrier.
7. A method for synthesizing bivalirudin, which comprises coupling Fmoc-Leu-OH with the spherical carrier of claim 1 as a liquid phase synthesis carrier, removing amino protecting group, coupling Fmoc-Tyr (tBu) -OH, Fmoc-Glu (OtBu) -OH, Fmoc-Pro-OH, Fmoc-Ile-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Phe-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Gly-OH, Fmoc-Asn- (trt) -OH, Fmoc-Gly-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg (Of) -OH, Fmoc-Pro-OH, Pboc-Pro-OH, Fmoc-Ile-, Boc-D-Phe-OH, removing protective group, synthesizing the carrier by lysate phase, and purifying to obtain bivalirudin.
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Citations (4)
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CN102286076B (en) * | 2011-06-23 | 2014-03-12 | 成都圣诺科技发展有限公司 | Preparation method for bivalirudin |
CN104478995A (en) * | 2014-12-30 | 2015-04-01 | 郑州大学 | Method for synthetizing fullerene bis-addition polypeptide by combining liquid phase and solid phase |
CN104478994A (en) * | 2014-12-30 | 2015-04-01 | 郑州大学 | Fullerene single-addition line peptide as well as preparation method and application thereof |
CN106083626A (en) * | 2016-06-03 | 2016-11-09 | 厦门大学 | One fullerene amino-acid ester self assembly carried medicine sustained-release vesicle material and its preparation method and application |
-
2017
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Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
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CN102286076B (en) * | 2011-06-23 | 2014-03-12 | 成都圣诺科技发展有限公司 | Preparation method for bivalirudin |
CN104478995A (en) * | 2014-12-30 | 2015-04-01 | 郑州大学 | Method for synthetizing fullerene bis-addition polypeptide by combining liquid phase and solid phase |
CN104478994A (en) * | 2014-12-30 | 2015-04-01 | 郑州大学 | Fullerene single-addition line peptide as well as preparation method and application thereof |
CN106083626A (en) * | 2016-06-03 | 2016-11-09 | 厦门大学 | One fullerene amino-acid ester self assembly carried medicine sustained-release vesicle material and its preparation method and application |
Non-Patent Citations (3)
Title |
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Arylation of [60]fullerene via electrophilic aromatic substitution involving the electrophile C60Cl6: frontside nucleophilic substitution of fullerenes;BIRKETT, P.R.等;《J. Chem. Soc., Perkin Trans. 2》;19970101;第6卷;第2783-2791页 * |
Carboxylic fullerene C60 derivatives: efficient microbicides against herpes simplex virus and cytomegalovirus infections in vitro;FEDOROVA, N.E.等;《Mendeleev Commun.》;20121231;第22卷;第254-256页 * |
Chlorofullerene C60Cl6: a precursor for straightforward preparation of highly water-soluble polycarboxylic fullerene derivatives active against HIV;TROSHINA, O.A.等;《Org. Biomol. Chem.》;20070718;第5卷;第1121-1125页 * |
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