CN118239872A - Acrylamide modified cysteine and synthesis and application thereof - Google Patents

Acrylamide modified cysteine and synthesis and application thereof Download PDF

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CN118239872A
CN118239872A CN202311340910.XA CN202311340910A CN118239872A CN 118239872 A CN118239872 A CN 118239872A CN 202311340910 A CN202311340910 A CN 202311340910A CN 118239872 A CN118239872 A CN 118239872A
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cysteine
allyloxycarbonyl
polypeptide
acrylamide
reaction
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孙浩
邵佳琦
宋晶林
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Kangming Yongrui Biotechnology Suzhou Co ltd
Nanjing Agricultural University
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Kangming Yongrui Biotechnology Suzhou Co ltd
Nanjing Agricultural University
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Abstract

The invention discloses an acrylamide modified cysteine and synthesis and application thereof, and the technical scheme for synthesizing the molecule comprises the following steps: firstly, nitrogen methylol acrylamide reacts with cysteine under the action of acid, and the obtained acrylamide modified cysteine reacts with allyl chloroformate under the action of alkali, so that the amino group of the cysteine is protected, and finally the acrylamide modified allyloxycarbonyl cysteine is obtained. The special cysteine molecule synthesized by the invention can be used for solid-phase polypeptide synthesis and can be introduced into a polypeptide sequence as special amino acid. Subsequently, using the more reactive acrylamide double bond in the molecule, various tag units can be introduced, including the polypeptide solubilizing tag (Arg) n. The tag unit introduced by the cysteine side chain and the acrylamide group can be removed together by palladium chloride in an aqueous solution to obtain a natural polypeptide sequence.

Description

Acrylamide modified cysteine and synthesis and application thereof
Technical Field
The invention belongs to the technical field of organic synthesis, and particularly relates to acrylamide modified cysteine and synthesis and application thereof.
Background
The introduction of tag unit modifications (e.g., solubilizing tags) during solid phase polypeptide synthesis by synthesis of specific amino acids has become an important method for polypeptide modification. In 2013, liu Keti groups invented a special glutamine (Gln) side chain modified by synthesized o-nitrobenzyl (Arg) n solubilizing-aid tag unit, and successfully synthesized LC3-II(Huang,Y.-C.;Li,Y.-M.;Chen,Y.;Pan,M.;Li,Y.-T.;Yu,L.;Guo,Q.-X.;Liu,L.Synthesis of Autophagosomal Marker Protein LC3-II under Detergent-Free Conditions.Angewandte Chemie International Edition 2013,52(18),4858-4862.), by using Fmoc-Glu-OMe as a raw material and synthesizing the o-nitrobenzyl modified glutamine target molecule through three steps, wherein the nitrobenzyl and the tag unit can be removed together under the condition of illumination. In 2014, liu Keti groups invented a method for introducing a tag unit by synthesizing special glycine, namely, a commercial 2, 4-dihydroxybenzaldehyde is synthesized into an Fmoc-Gly RMB0 -OH with an Hmb group through seven steps of reactions, the existence of N-methyl-N- [2- (methylamino) -ethyl ] -carbamoyl of the Hmb group enables the special glycine to exist stably in polypeptide solid-phase synthesis, the Hmb group and the tag unit can be removed by TFA together for (Zheng,J.-S.;Yu,M.;Qi,Y.-K.;Tang,S.;Shen,F.;Wang,Z.-P.;Xiao,L.;Zhang,L.;Tian,C.-L.;Liu,L.Expedient total synthesis of small to medium-sized membrane proteins via Fmoc chemistry.J Am Chem Soc 2014,136(9),3695-3704.).2016 years, brik subject group takes 4-aminobenzonitrile as a raw material, fmoc-Cys (PhacmNHAlloc) -OH is synthesized through four steps of reactions, an (Arg) n tag unit is introduced into a side chain of cysteine (Cys), a successful synthesis strategy for H4 protein (Maity,S.K.;Mann,G.;Jbara,M.;Laps,S.;Kamnesky,G.;Brik,A.Palladium-Assisted Removal of a Solubilizing Tag from a Cys Side Chain To Facilitate Peptide and Protein Synthesis.Org Lett 2016,18(12),3026-3029.), comprises protecting an amino group of 4-aminophenylacetonitrile with allyloxycarbonyl, then hydrolyzing a nitrile functional group into an amide through acid catalysis, then converting the amide into a azamethylol amide group, then reacting with Fmoc-Cys-OH under acidic conditions to obtain phenylacetylaminomethyl-modified cysteine, and the palladium acetyl methyl-modified cysteine can be removed by chlorination together with the tag unit. In 2018, danishefasky group introduced tag unit by special cysteine (Fmoc-Cys (AcmNHAlloc) -OH) synthesized in two steps, acetamidomethyl bond and tag molecule can be removed together by palladium (Brailsford,J.A.;Stockdill,J.L.;Axelrod,A.J.;Peterson,M.T.;Vadola,P.A.;Johnston,E.V.;Danishefsky,S.J.Total chemical synthesis of human thyroid-stimulating hormone(hTSH)β-subunit:Application of arginine-tagged acetamidomethyl(AcmR)protecting groups.Tetrahedron 2018,74(15),1951-1956).
Although the above-mentioned special amino acid of the invention has been successfully applied to the synthetic modification of difficult proteins, there are some limitations in the research in this field, on the one hand, the synthetic procedure of the special amino acid for introducing a tag unit is complicated, the synthetic cost is high, and there is a limitation in wide application. On the other hand, the introduction of the tag unit has strong tolerance and stability under various reaction conditions of protein synthesis, and can be conveniently and efficiently removed without side reaction. This requires that we propose new breakthroughs and innovations in strategies for introducing tag unit modifications, applied to the chemical synthesis, modification, characterization and analysis of more extensive difficult proteins.
Disclosure of Invention
The invention is based on the prior art, and aims to provide acrylamide modified cysteine and synthesis and application thereof; the modified cysteines of the invention can be used to introduce a desired tag unit in solid phase polypeptide synthesis.
The technical scheme adopted by the invention is as follows:
In a first aspect, the invention discloses an acrylamide modified allyloxycarbonyl cysteine having the formula: alloc-Cys (CH 2NHCOCH＝CH2) -OH, having the structural formula:
In a second aspect, the invention discloses a method for synthesizing acrylamide modified allyloxycarbonyl cysteine, which comprises the steps of respectively modifying amino and sulfhydryl groups of cysteine by using allyl chloroformate and azoxymethacrylamide through two-step reaction to obtain acrylamide modified allyloxycarbonyl cysteine; mainly selected from the following (A1) or (A2):
(A1) Firstly, reacting cysteine with azoxymethacrylamide under an acidic condition to obtain acrylamide modified cysteine, and then reacting with allyl chloroformate under an alkaline condition to obtain a target molecule;
(A2) Firstly, under alkaline condition, cystine and allyl chloroformate react to obtain allyloxycarbonyl cystine, and then the allyloxycarbonyl cystine reacts with nitrogen methylol acrylamide under the action of a reducing agent and acid to obtain a target molecule.
In a specific embodiment, in the (A1), the molar ratio of cysteine to azoxymethacrylamide is 1:1-3, and may be 1:1. 1:2 or 1:3, etc., the reaction temperature is room temperature.
In a specific embodiment, in the (A1), the molar ratio of cysteine to allyl chloroformate to base is: 1:1 to 3:1 to 3, may be 1:1: 1. 1:2: 2. 1:3:3, and the like, wherein the reaction conditions are room temperature and nitrogen atmosphere.
In a specific embodiment, in the (A2), the molar ratio of cystine to allyl chloroformate to base is 1:2 to 6:2 to 6; can be 1:2: 2. 1:4: 4. or 1:6:6, and the like, wherein the reaction conditions are room temperature and nitrogen atmosphere.
In a specific embodiment, in the (A2), the molar ratio of cystine to reducing agent and azoxymethacrylamide is 1:1 to 3:2 to 6; can be 1:1: 2. 1:2: 4. or 1:3:6, etc., the reaction conditions are room temperature.
In a specific embodiment, the reaction solvent is selected from any one of dichloromethane, acetonitrile, tetrahydrofuran, N-methylpyrrolidone, N-dimethylformamide.
In a specific embodiment, the acid reagent in acidic condition is selected from any one of trifluoroacetic acid, difluoroacetic acid, acetic acid, formic acid, hydrochloric acid, trichloroacetic acid.
In a specific embodiment, the alkaline reagent under alkaline conditions is selected from any one of sodium carbonate, sodium bicarbonate, sodium hydroxide, triethylamine, diisopropylethylamine.
In a third aspect, the invention also provides the use of an acrylamide modified allyloxycarbonyl cysteine as hereinbefore described in polypeptide synthesis.
In a fourth aspect, the present invention also provides a method for synthesizing a polypeptide, comprising the steps of:
(1) Preparing the acrylamide modified allyloxycarbonyl cysteine described above;
(2) Introducing acrylamide modified allyloxycarbonyl cysteine into the polypeptide fragment by solid phase polypeptide synthesis;
(3) Introducing piperazine into the polypeptide fragment through an acrylamide double bond;
(4) Introducing a tag unit to the polypeptide fragment via piperazine;
(5) Removing an allyloxycarbonyl protecting group of an amino group on cysteine;
(6) Continuing solid-phase polypeptide synthesis to obtain linear peptide resin;
(7) Cracking and freeze-drying polypeptide resin to obtain crude peptide;
(8) And removing cysteine side chain modification units by using palladium chloride to obtain the natural polypeptide.
In a specific embodiment, the condensing agent used in step (2), step (4) and step (6) is one or more of the following condensing agents: DIC, HATU, TBTU, HBTU, pyBop, HOBt, cl-HOBt, DIPEA, NMM, DIEA, oxyma.
In a specific embodiment, the solvent used for introducing piperazine in step (3) is selected from any one of dichloromethane, N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, N-methylmorpholine, acetonitrile.
In a specific embodiment, the piperazine solution concentration in step (3) is from 0.5M to 1M.
In a specific embodiment, the reagent for removing allyloxycarbonyl in step (5) and the corresponding ratio are: pd (PPh 3)4: phenylsilane=0.2 equiv.:20 equiv.: the solvent is anhydrous dichloromethane.
In a specific embodiment, the cleavage liquid in step (7) is TFA: TIS: h 2 o=95%: 2.5%:2.5% (V: V: V) and pouring out the supernatant after low-temperature centrifugation in methyl tert-butyl ether, and obtaining the crude peptide by precipitation, dissolution and freeze-drying.
In a specific embodiment, the reagents and reaction conditions for removing cysteine side chain modifying units in step (8) are: palladium chloride (100 mM) was dissolved in 6M Gn.HCl/200 mM PBS (pH=7.0) and the reaction temperature was 37 ℃.
In a fifth aspect, the invention also provides polypeptides synthesized by the synthesis methods described above.
Advantageous effects
The invention designs and synthesizes the allyloxycarbonyl cysteine modified by acrylamide, has novel molecular design structure, and can be used as special amino acid for polypeptide solid phase synthesis. The raw materials required for synthesizing the molecule are L-cysteine/cystine, azoxymethacrylamide and allyl chloroformate, and the molecule is simple and easy to obtain and has low cost. The synthesis process is two-step organic reaction, and the product with the purity of more than 98 percent can be obtained through one-time silica gel column separation, and the yield is 67.4 percent. The special amino acid can be used for introducing a tag unit in solid-phase polypeptide synthesis to realize synthesis of hydrophobic polypeptide and site-directed modification of polypeptide.
Drawings
FIG. 1 is a graph showing the results of HPLC and Mass detection of polypeptide fragment 1;
FIG. 2 is a graph showing the results of HPLC and Mass detection of polypeptide fragment 3;
FIG. 3 is a graph showing the results of HPLC and Mass detection of polypeptide fragment 4;
FIG. 4 is a graph showing the results of HPLC and Mass detection of polypeptide fragment 5;
FIG. 5 is a graph showing HPLC and Mass detection results of purified polypeptide fragment 6;
FIG. 6 is a graph showing the results of HPLC and Mass detection of polypeptide fragment 7;
FIG. 7 is a nuclear magnetic resonance spectrum of acrylamide modified allyloxycarbonyl cysteine (Alloc-Cys (CH 2NHCOCH＝CH2) -OH);
FIG. 8 is a nuclear magnetic carbon spectrum of acrylamide modified allyloxycarbonyl cysteine (Alloc-Cys (CH 2NHCOCH＝CH2) -OH);
FIG. 9 is a mass spectrum of acrylamide modified allyloxycarbonyl cysteine (Alloc-Cys (CH 2NHCOCH＝CH2) -OH).
Detailed Description
The present invention is described in detail below in conjunction with the following examples, which are based on the technical solutions of the present invention and provide detailed embodiments and specific operation procedures, the scope of the present invention includes but is not limited to the following examples.
1. Synthesis of acrylamide-modified allyloxycarbonyl cysteine
Example 1
L-cysteine (10 mmol,1.21 g) and azoxymethacrylamide (2.0 equiv.,20mmol,2.02 g) were placed in a round bottom flask, 10mL dichloromethane and 10mL trifluoroacetic acid were added, the system was left to stir at room temperature to react, TLC monitored for reaction completion, and concentrated under reduced pressure to give a pale yellow transparent oil, the crude product was used directly in the next step without purification.
The first crude product was dissolved with 8mL of acetonitrile and 14mL of water, the reaction mixture was brought to neutral with sodium carbonate, then sodium carbonate (2.0 equiv.,20mmol,2.12 g) was added, and allyl chloroformate (2.0 equiv.,20mmol,2.12 mL) was added at 0 ℃ under nitrogen protection, then the reaction was monitored by stirring TLC at room temperature, the reaction was completed for 18 hours, and the reaction mixture was brought to acidity with 5% aqueous hydrochloric acid. The solution was washed three times with ethyl acetate, the organic phase was collected and dried over anhydrous sodium sulfate, filtered, and the crude product obtained by concentrating the filtrate under reduced pressure was purified by column chromatography (dichloromethane: methanol=4:1). The reaction was carried out in two steps to give 1.94g (yield: 67.4%) of the desired product.
Nuclear magnetism and mass spectrum data of target product ：1H NMR(500MHz,DMSO-d6)δ8.85-8.65(m,1H),7.38(s,1H),6.31-6.15(m,1H),6.15-6.03(m,1H),5.89(m,1H),5.63(ddd,J＝9.5,6.5,3.3Hz,1H),5.37-5.22(m,1H),5.18(d,J＝10.9Hz,1H),4.47(t,J＝6.1Hz,2H),4.32(m,2H),4.11(td,J＝10.0,9.4,4.9Hz,1H),3.03(s,1H),2.80(dd,J＝13.5,9.3Hz,1H),1.37-1.12(m,1H).13C NMR(125.8MHz,DMSO-d6)δ173.18,165.46,156.28,134.07,131.74,126.62,117.57,64.99,54.91,41.04,32.94.MS(ESI):calculated for C11H16N2O5S[M+H]+:289.08,found:289.08
Example 2
L-cysteine (10 mmol,1.21 g) and azoxymethacrylamide (2.0 equiv.,20mmol,2.02 g) were placed in a round bottom flask, 10mL dichloromethane and 10mL trifluoroacetic acid were added, the system was left to stir at room temperature to react, TLC monitored for reaction completion, and concentrated under reduced pressure to give a pale yellow transparent oil, the crude product was used directly in the next step without purification.
The first crude product was dissolved with 8mL acetonitrile and 14mL water, the pH of the mixture was adjusted to neutral with sodium bicarbonate, then sodium bicarbonate (2.0 equiv.,20mmol,1.68 g) was added, and allyl chloroformate (2.0 equiv.,20mmol,2.12 mL) was added at 0 ℃ under nitrogen protection, then the system was left to stir at room temperature for reaction, TLC monitored for reaction, and the pH of the reaction solution was adjusted to acidic with 5% aqueous hydrochloric acid after completion of the reaction for 18 hours. The solution was washed three times with ethyl acetate, and the organic phase was collected and dried over anhydrous sodium sulfate, filtered, and the crude product obtained by concentrating the filtrate under reduced pressure was purified by column chromatography (dichloromethane: methanol=4:1) to give 0.81g of the objective product (yield: 28.1%).
Example 3
L-cystine (5 mmol,1.20 g) and sodium carbonate (2.0 equiv.,10mmol,1.06 g) were placed in a round bottom flask, nitrogen was purged, 4mL acetonitrile and 8mL water were added, allyl chloroformate (4.0 equiv.,20mmol,2.12 mL) was added at 0deg.C, then the system was left to stir at room temperature to react, TLC monitored for reaction, and the reaction was quenched by 5% aqueous hydrochloric acid to pH acidity. The solution was washed three times with ethyl acetate, and the organic phase was collected and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product for the next step.
To the resulting crude product was added tricarboxyethyl phosphine (2.0 equiv.,10mmol,2.86 g), azoxymethyl acrylamide (4.0 equiv.,20mmol,2.02 g), 10mL dichloromethane and 10mL trifluoroacetic acid, the system was left to stir at room temperature, the reaction was monitored by TLC, the reaction was completed for 4 hours, concentrated under reduced pressure, the organic phase was extracted with water and dichloromethane, the organic phase extract was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the crude product, which was purified by column chromatography (dichloromethane: methanol=4:1). 1.20g of the target product was obtained (yield: 41.8%).
2. The dissolution-aiding tag unit is introduced into the polypeptide through the special cysteine (Alloc-Cys (CH 2NHCOCH＝CH2) -OH)
2.1 Introduction of acrylamide-modified allyloxycarbonyl cysteine into polypeptide fragments by solid phase polypeptide Synthesis
RINK AMIDE-MBHA Resin (0.37 mmol/g,0.1 mmol) was weighed and added to a solid phase reaction column, washed 2 times with N, N-dimethylformamide and the Resin swollen with N, N-dimethylformamide for 30min. Deprotection was carried out three times by adding 20% piperidine for 3 minutes, 4 minutes and 3 minutes respectively, and the resin was washed 6 times with N, N-dimethylformamide. Fmoc-Gly-OH (5.0 equiv.,0.5mmol,148.7 mg) and ethyl 2-oxime cyanoacetate (5.0 equiv.,0.5mmol,71 mg) were weighed, dissolved in 1mL of N, N-dimethylformamide, diisopropylcarbodiimide (6.0 equiv.,0.6mmol, 93. Mu.L) was added, activated for 3min, and then added to the resin, reacted at room temperature for 1h, and the reaction was terminated with ninhydrin (termination of the reaction if the resin was colorless and transparent; extension of the reaction for 1h if the resin was developed). After the reaction, the reaction mixture was removed, and the resin was washed three times with N, N-dimethylformamide.
According to the first step of reaction, the coupling Fmoc-Ala-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Leu-OH、Alloc-Cys(CH2NHCOCH＝CH2)-OH, is carried out for 1h, and the ninhydrin is used for detecting the reaction end point (if the resin is colorless and transparent, the reaction is stopped, and if the resin is colored, the reaction is prolonged for 1 h). The analysis method adopts the following high performance liquid chromatography conditions: mobile phase: a:0.1% TFA/H 2 O, B:0.1% TFA/ACN; chromatographic column: XBridge (waters, BEH C18,3.5 μm,4.6 Mm. Times.150 mm); flow rate: 1.2mL/min; the procedure is as follows: 0-60% B; time: 30min; detection wavelength: 214nm. The RP-HPLC and ESI-MS detection results are shown in FIG. 1.
2.2 Introduction of piperazine and solubilizing tag units into polypeptide fragments by acrylamide double bond
2G of piperazine was weighed, dissolved in 30mL of N, N-dimethylformamide, and added to a solid phase reaction column to react for 2 hours at room temperature. Fmoc-Arg (Pbf) -OH, fmoc-Arg (Pbf) -OH, boc-Arg (Pbf) -OH, and then sequentially coupled to piperazine according to the first reaction step described above. The RP-HPLC and ESI-MS detection results are shown in FIG. 2.
2.3 Removal of the allyloxycarbonyl protecting group from the amino group of cysteine in the resin polypeptide
Pd (PPh 3)4 (0.2 equiv.,0.02mmol,23 mg), phenylsilane (20 equiv.,2mmol, 246. Mu.L) was dissolved in 3mL of anhydrous dichloromethane, the solvent was poured into the dried resin, and the reaction was carried out at room temperature for 1h.
2.4 Continuous solid phase polypeptide Synthesis to Linear peptide resin
Fmoc-Gly-OH, fmoc-Ala-OH, fmoc-Arg (Pbf) -OH, fmoc-Tyr (tBu) -OH and Fmoc-Leu-OH were coupled sequentially according to the 2.1 reaction steps, and reacted at room temperature for 1h, and the end point of the reaction was detected with ninhydrin (if the resin was colorless and transparent, the reaction was terminated; if the resin was developed, the reaction was prolonged for 1 h). The RP-HPLC and ESI-MS detection results are shown in FIG. 4.
2.5 Cracking and freeze-drying the polypeptide resin to obtain crude peptide.
The resin was washed 3 times with N, N-dimethylformamide, 2 times with dichloromethane, dried under vacuum, 5mL of the pre-prepared lysate was added, reacted at room temperature for 2h, the resin was filtered, the filtrate was collected, the resin was washed with 1mL of the lysate, and the filtrates were combined. Slowly adding the filtrate into 35mL of glacial ethyl ether for precipitation, centrifuging, discarding supernatant, lyophilizing to obtain crude peptide, and performing liquid phase separation and purification to obtain pure polypeptide (compound 6). The purification method adopts the preparation high performance liquid chromatography, and the conditions are as follows: mobile phase: a:0.1% TFA/H 2 O, B:0.1% TFA/ACN; chromatographic column: XBridge (waters, BEH C18, 10 μm,19Mm by 250 mm); flow rate: 12mL/min, detection wavelength: 214nm. The RP-HPLC and ESI-MS detection results are shown in FIG. 5.
2.6 Removal of cysteine side chain modification units Using Palladium chloride to obtain Natural Polypeptides
Weighing PdCl 2 1.8.8 mg, dissolving in 100 mu L of 6M Gn.HCl/200 mM PBS buffer solution, and incubating the mixture at 37 ℃ for 10min for dissolution; 0.25mg of the purified peptide was weighed and dissolved in 60. Mu.L of 6M Gn.HCl/200 mM PBS solution, and 14. Mu.L of PdCl 2 solution was added thereto to react at 37℃for 10 minutes. The reaction mixture was quenched by adding an excessive amount of dithiothreitol, centrifuged, and the supernatant was analyzed and detected by RP-HPLC and ESI-MS, and the results are shown in FIG. 6.
The english abbreviations and chinese names of the main raw materials used in the synthesis of the above-mentioned polypeptide sequences correspond to those shown in table 1 below:
TABLE 1 substance names corresponding to polypeptide amino acids and abbreviations for synthetic raw materials
While the preferred embodiments of the present application have been described in detail, the present application is not limited to the embodiments, and various equivalent modifications and substitutions can be made by those skilled in the art without departing from the spirit of the present application, and these equivalent modifications and substitutions are intended to be included in the scope of the present application as defined in the appended claims.

Claims (10)

1. An acrylamide modified allyloxycarbonyl cysteine having the structural formula:
2. The method for synthesizing the acrylamide modified allyloxycarbonyl cysteine according to claim 1, wherein the method comprises the steps of: modifying amino and sulfhydryl of cysteine by allyl chloroformate and azoxymethacrylamide through two-step reaction to obtain acrylamide modified allyloxycarbonyl cysteine; mainly selected from the following (A1) or (A2):
(A1) Firstly, reacting cysteine with azoxymethacrylamide under an acidic condition to obtain acrylamide modified cysteine, and then reacting with allyl chloroformate under an alkaline condition to obtain a target molecule;
(A2) Firstly, under alkaline condition, cystine and allyl chloroformate react to obtain allyloxycarbonyl cystine, and then the allyloxycarbonyl cystine reacts with nitrogen methylol acrylamide under the action of a reducing agent and acid to obtain a target molecule.
3. The synthesis method according to claim 2, wherein in (A1), the molar ratio of cysteine to azoxymethacrylamide is 1:1-3, preferably 1:2, the reaction temperature is room temperature;
preferably, the molar ratio of cysteine to allyl chloroformate to base is: 1:1 to 3:1 to 3, preferably 1:2:2, the reaction condition is room temperature and nitrogen atmosphere.
4. The method according to claim 2, wherein in (A2), the molar ratio of cystine to allyl chloroformate to base is 1:2 to 6:2 to 6; preferably 1:4:4, the reaction condition is room temperature and nitrogen atmosphere;
preferably, the molar ratio of cystine to reducing agent and azoxymethacrylamide is 1:1 to 3:2 to 6; preferably 1:2:4, the reaction condition is room temperature.
5. The synthetic method according to claim 2, wherein the reaction solvent is selected from any one of dichloromethane, acetonitrile, tetrahydrofuran, N-methylpyrrolidone, N-dimethylformamide; the acid reagent is any one of trifluoroacetic acid, difluoroacetic acid, acetic acid, formic acid, hydrochloric acid and trichloroacetic acid; the alkali reagent is selected from any one of sodium carbonate, sodium bicarbonate, sodium hydroxide, triethylamine and diisopropylethylamine.
6. Use of an acrylamide modified allyloxycarbonyl cysteine of claim 1 in polypeptide synthesis.
7. A method for synthesizing a polypeptide, comprising the steps of:
(1) Modifying amino and sulfhydryl of cysteine by allyl chloroformate and azoxymethacrylamide through two-step reaction to obtain acrylamide modified allyloxycarbonyl cysteine;
(2) Introducing acrylamide modified allyloxycarbonyl cysteine into the polypeptide fragment by solid phase polypeptide synthesis;
(3) Introducing piperazine into the polypeptide fragment through an acrylamide double bond;
(4) Introducing a tag unit to the polypeptide fragment via piperazine;
(5) Removing an allyloxycarbonyl protecting group of an amino group on cysteine;
(6) Continuing solid-phase polypeptide synthesis to obtain linear peptide resin;
(7) Cracking and freeze-drying polypeptide resin to obtain crude peptide;
(8) And removing cysteine side chain modification units by using palladium chloride to obtain the natural polypeptide.
8. The method of claim 7, wherein the step of determining the position of the probe is performed,
In the step (2), the step (4) and the step (6), the condensing agent is one or more of the following condensing agents: DIC, HATU, TBTU, HBTU, pyBop, HOBt, cl-HOBt, DIPEA, NMM, DIEA, oxyma; preferably, the solvent used for introducing piperazine in the step (3) is selected from any one of dichloromethane, N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, N-methylmorpholine and acetonitrile;
Preferably, the piperazine solution concentration in step (3) is 0.5M to 1M.
9. The method of claim 7, wherein the step of determining the position of the probe is performed,
The reagent for removing allyloxycarbonyl in the step (5) and the corresponding proportion are as follows: pd (PPh 3)4: phenylsilane=0.2 equiv.:20 equiv.;
preferably, the cleavage liquid in step (7) is TFA: TIS: h 2 o=95%: 2.5%:2.5% (V: V: V) of centrifuging in methyl tertiary butyl ether at low temperature, pouring out supernatant, precipitating, dissolving and freeze-drying to obtain crude peptide;
preferably, the reagent for removing cysteine side chain modification unit in step (8) and the reaction conditions are as follows: 100mM palladium chloride was dissolved in 6 MGn.HCl/200 mM PBS, pH=7.0, and the reaction temperature was 37 ℃.
10. A polypeptide synthesized by the synthetic method of any one of claims 7-9.
CN202311340910.XA 2023-10-16 Acrylamide modified cysteine and synthesis and application thereof Pending CN118239872A (en)

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