CN112940071B - Method for realizing alkynyl functionalization of cysteine and polypeptide thereof by utilizing microchannel reactor - Google Patents
Method for realizing alkynyl functionalization of cysteine and polypeptide thereof by utilizing microchannel reactor Download PDFInfo
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- C07K1/1072—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 by covalent attachment of residues or functional groups
- C07K1/1077—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 by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
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- C07K5/06—Dipeptides
- C07K5/06139—Dipeptides with the first amino acid being heterocyclic
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Abstract
The invention discloses a method for realizing alkynyl functionalization of cysteine and polypeptides thereof by utilizing a microchannel reactor, which comprises the following steps: (1) Dissolving cysteine or polypeptide containing cysteine shown in formula I in a first solvent to obtain a first reaction solution; (2) Dissolving an alkynyl functionalization reagent in a second solvent to serve as a second reaction solution; (3) Pumping the first reaction liquid and the second reaction liquid into a micro-channel reactor respectively and simultaneously for reaction, and collecting effluent liquid to obtain the reaction liquid containing cysteine shown in the formula II or the alkynyl functional product of the polypeptide containing cysteine. The invention relates to a brand-new method for realizing alkynyl functional modification of cysteine and polypeptide thereof, which can realize alkynyl functional modification of cysteine and polypeptide thereof only by adding organic alkali into a reaction system.
Description
Technical Field
The invention belongs to the field of chemical synthesis, and particularly relates to a method for realizing alkynyl functionalization of cysteine and polypeptides thereof by utilizing a microchannel reactor.
Background
Cysteine is one of 20 common amino acids that has important applications in a number of fields, for example: cysteine can be used as a raw material for synthesizing glutathione in a living body, and the glutathione is an important antioxidant substance in the living body; cysteine also has applications in the fields of pharmaceutical molecules and food processing; in addition, because cysteine contains a sulfhydryl structure, quantitative detection of the sulfhydryl content in a living body can be used as a judgment basis for related diseases, and the cysteine is also an important research object for scientific researchers.
Currently, methods for realizing alkynyl functional modification of cysteine and polypeptides thereof by using a micro-flow field reaction technology are also recently reported, and a method for realizing amino acid arylation by using aniline and cysteine under an illumination condition is reported by Timothy Noel in 2017 (Angew.chem.int.ed.2017, 56, 12702-12707). Although the reaction can effectively realize the arylation of amino acid, a photocatalyst and an oxidant are added into a reaction system. In 2018, veronique Gouverneur reported a method for performing difluoromethylation modification of cysteine-containing polypeptides using Umemoto reagent (j.am. Chem. Soc.2018,140, 1572-1575). Although the method has a good substrate range, the reaction yield is low, the synthesis scale is small, and the research of a scale-up experiment cannot be carried out. The traditional method for functionally modifying the cysteine often has the defects of needing to use noble metal as a catalyst, having low atom utilization rate, being not friendly to the environment and the like, and the defects limit the application of the method in industrialization. Therefore, it is very interesting to develop a catalyst-free, mild reaction conditions, environmentally friendly and easily scalable method for cysteine functionalization modification.
Disclosure of Invention
The invention aims to: the invention aims to solve the technical problem of providing a method for realizing alkynyl functionalization of cysteine and polypeptides thereof by utilizing a microchannel reactor aiming at the defects of the prior art.
In order to solve the technical problems, the invention discloses a method for realizing alkynyl functionalization of cysteine and polypeptides thereof by utilizing a microchannel reactor, as shown in fig. 1, comprising the following steps:
(1) Dissolving cysteine or polypeptide containing cysteine shown in formula I in a first solvent to obtain a first reaction solution;
(2) Dissolving an alkynyl functionalization reagent in a second solvent to serve as a second reaction solution;
(3) Pumping the first reaction liquid and the second reaction liquid into a micro-channel reactor respectively and simultaneously for reaction, and collecting effluent liquid to obtain a reaction liquid containing cysteine shown in a formula II or an alkynyl functional product of the polypeptide containing cysteine;
wherein, the liquid crystal display device comprises a liquid crystal display device,
R 1 selected from hydrogen, methyl, ethyl, propyl or isopropyl, preferably methyl;
R 2 selected from-Ac (acetyl), -Boc (t-butoxycarbonyl), -Cbz (benzyloxycarbonyl), -Ts (p-toluenesulfonyl), -Fmoc (fluorenylmethoxycarbonyl) or other cysteine-linked amino acids, preferably other cysteine-linked amino acids; further preferably, the compound is any one of structures represented by formula IV (containing cysteine);
preferably, the cysteine or cysteine-containing polypeptide of formula I is any one of the structures of formula V:
R 3 selected from alkanes, cycloalkanes, aryl derivatives or heterocyclic structures, preferably aryl or aryl derivatives, further preferably phenyl, 4-trifluoromethylphenyl, 4-tert-butylphenylTIPS (triisopropylsilyl), naphthalene.
Wherein the concentration of cysteine or cysteine-containing polypeptide in the first solution is 0.05-1.0 mmol/mL, preferably 0.05-0.5 mmol/mL.
Preferably, the first reaction solution further includes an organic base.
Wherein the organic base includes, but is not limited to, pyridine, 2, 6-lutidine, 2, 6-di-tert-butylpyridine, N, N-diisopropylethylamine, triethylenediamine, N, N, N ', N' -tetramethyl ethylenediamine, 4-dimethylaminopyridine, triethylamine.
Wherein, in the first solution, the mole ratio of cysteine or polypeptide containing cysteine to organic alkali is 1:1 to 5, preferably 1:1.5.
wherein the alkynyl functionalization reagent is a compound shown in a formula III;
wherein R is 3 Selected from alkanes, cycloalkanes, aryl derivatives or heterocyclic structures, preferably aryl or aryl derivatives, more preferably phenyl, 4-trifluoromethylphenyl, 4-tert-butylphenyl, TIPS (triisopropylsilyl), naphthalene.
Wherein, in the second solution, the concentration of the alkynyl functionalization reagent is 0.05-2.0 mmol/mL, and is preferably 0.15-1.0 mmol/mL.
Wherein the molar ratio of the cysteine or the polypeptide containing the cysteine to the alkynyl functionalization reagent is 1:1-5.
Wherein the first solvent and the second solvent are respectively and independently selected from methanol, ethanol, acetone, 1, 4-dioxane, dichloromethane, 1, 2-dichloroethane, N-dimethyl propenyl urea, acetonitrile, N-dimethylformamide, N-dimethylacetamide, water, phosphate buffer, tetrahydrofuran, dimethyl sulfoxide or any combination thereof, and preferably dimethyl sulfoxide.
Wherein the pumping rate of the first solution and the second solution is 1:1.
wherein the micro-channel reactor comprises a feed pump (Baoding Leifu Fluid Technology Co.Ltd, TYD01-01-CE type), a mixing module (with an inner diameter of 0.6 mm), a micro-reactor and a receiver; wherein, the reaction liquid pumped by the feed pump flows into the microreactor for reaction after being mixed by the mixing module, and the reaction is shown in figure 2.
Wherein the mixing module is a Y-shaped or T-shaped mixer.
Wherein the microreactor is of a pore canal structure, the pore canal material is Perfluoroalkoxyalkane (PFA) or polytetrafluoroethylene, the size and the inner diameter of the microreactor are 0.5-1.0 mm, the length is 5-20 m, and the volume is 1-15.7 mL; wherein the inner diameter is preferably 0.6mm, the volume is preferably 1.4mL, and the flow rate is 0.1-2.0 mL/min.
Wherein the temperature of the reaction is room temperature.
Wherein the reaction time is 30s to 2.6 hours, preferably 1min to 60min, more preferably 1min to 30min, still more preferably 1min to 10min, and most preferably 4.7min.
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
(1) The invention relates to a brand-new method for realizing alkynyl functional modification of cysteine and polypeptide thereof, which can realize alkynyl functional modification of cysteine and polypeptide thereof only by adding organic alkali into a reaction system.
(2) According to the invention, a catalyst is not required, and alkynyl functional modification of cysteine and polypeptide thereof can be realized under the condition of room temperature, so that the problem that a transition metal catalyst is required in the prior art is solved, and the reaction cost and the energy consumption cost are reduced.
(3) The system has the advantages of no solid insoluble matters, no microchannel blocking problem, simple operation, high safety, short reaction time, high reaction conversion rate and yield, and high reaction continuity, and is beneficial to continuous and uninterrupted amplified production, and the defects of the traditional method are overcome.
(4) The invention can realize the synthesis of polypeptide derivatives besides single amino acid derivatives.
(5) The raw material conversion rate of the invention is 88% -100%, and the product yield is as high as 83% -95%.
Drawings
The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.
FIG. 1 is a schematic illustration of the reaction scheme of the present invention.
FIG. 2 is a schematic diagram of a microchannel reactor apparatus.
FIG. 3 is a graph of N- ((tert-butoxycarbonyl) -L-phenylalanyl) -S- (phenylethynyl) -L-cysteine methyl ester hydrogen.
FIG. 4 is a chart of N- ((tert-butoxycarbonyl) -L-phenylalanyl) -S- (phenylethynyl) -L-cysteine methyl ester carbon.
FIG. 5 is a graph of N- ((tert-butoxycarbonyl) -L-tryptophan) -S- (phenylethynyl) -L-cysteine methyl ester hydrogen.
FIG. 6 is a chart of N- ((tert-butoxycarbonyl) -L-tryptophan) -S- (phenylethynyl) -L-cysteine methyl ester.
FIG. 7 is a graph of N- ((tert-butoxycarbonyl) -L-tyrosyl) -S- (phenylethynyl) -L-cysteine methyl ester hydrogen.
FIG. 8 is a carbon diagram of N- ((tert-butoxycarbonyl) -L-tyrosyl) -S- (phenylethynyl) -L-cysteine methyl ester.
FIG. 9 is a hydrogen spectrum of N- ((tert-butoxycarbonyl) -L-glutamyl) -S- (phenylethynyl) -L-cysteine methyl ester.
FIG. 10 is a chart of N- ((tert-butoxycarbonyl) -L-glutamyl) -S- (phenylethynyl) -L-cysteine methyl ester.
FIG. 11 is a hydrogen spectrum of N- (t-butoxycarbonyl) pentyl-L-prolyl-S- (phenylethynyl) -L-cysteine methyl ester.
FIG. 12 is a chart of N- (t-butoxycarbonyl) pentyl-L-prolyl-S- (phenylethynyl) -L-cysteine methyl ester carbon.
FIG. 13 is a graph of N- ((tert-butoxycarbonyl) -L-phenylalanyl) -S- (((4- (trifluoromethyl) phenyl) ethynyl) -L-cysteine methyl ester hydrogen.
FIG. 14 is a chart of N- ((tert-butoxycarbonyl) -L-phenylalanyl) -S- (((4- (trifluoromethyl) phenyl) ethynyl) -L-cysteine methyl ester carbon.
FIG. 15 is a fluorine spectrum of N- ((tert-butoxycarbonyl) -L-phenylalanyl) -S- (((4- (trifluoromethyl) phenyl) ethynyl) -L-cysteine methyl ester.
FIG. 16 is a graph of the hydrogen spectrum of methyl N- ((tert-butoxycarbonyl) -L-phenylalanyl) -S- (((4- (tert-butyl) phenyl) ethynyl) -L-cysteine.
FIG. 17 is a chart of N- ((tert-butoxycarbonyl) -L-phenylalanyl) -S- (((4- (tert-butyl) phenyl) ethynyl) -L-cysteine methyl ester carbon.
FIG. 18 is a graph of N- ((tert-butoxycarbonyl) -L-phenylalanyl) -S- ((triisopropylsilyl) ethynyl) -L-cysteine methyl ester hydrogen.
FIG. 19 is a chart of N- ((tert-butoxycarbonyl) -L-phenylalanyl) -S- ((triisopropylsilyl) ethynyl) -L-cysteine methyl ester.
FIG. 20 is a graph of N- ((tert-butoxycarbonyl) -L-phenylalanyl) -S- (naphthalen-1-ylethynyl) -L-cysteine methyl ester hydrogen.
FIG. 21 is a chart of N- ((tert-butoxycarbonyl) -L-phenylalanyl) -S- (naphthalen-1-ylethynyl) -L-cysteine methyl ester.
FIG. 22 is a graph of N-acetyl-S- (phenylethynyl) -L-cysteine methyl ester hydrogen.
FIG. 23 is a graph of N-acetyl-S- (phenylethynyl) -L-cysteine methyl ester carbon.
Detailed Description
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.
In the following examples, the flow rates of the first reaction liquid and the second reaction liquid are the same.
Example 1
(Boc) -L-phenylpropionyl-L-cysteine methyl ester 0.0764g (0.2 mmol,1.0 equiv.) was weighed out and dissolved in 2mTo L dimethyl sulfoxide was added 43. Mu.L triethylamine (0.3 mmol,1.5 equiv.) as a first reaction solution. 0.1308g of an alkynyl reagent (0.3 mmol,1.5 equiv.) was weighed out and dissolved in 2mL of dimethyl sulfoxide as a second reaction solution. The reaction solution was simultaneously pumped into a microreactor having an inner diameter of 0.6mm at a pumping flow rate of 0.15mL/min and a volume of 1.4mL, and the reaction residence time was 4.7min. After completion of the reaction, TLC was performed, the reaction solution was extracted with ethyl acetate and saturated brine (3X 25 mL), the organic layers were combined, dried over anhydrous sodium sulfate, and after the solvent was distilled off under reduced pressure, 86.7mg of the final product was obtained by silica gel column chromatography (petroleum ether: ethyl acetate=3:1), in 90% yield. Characterization data were as follows (fig. 3, fig. 4): 1 H NMR(400MHz,Chloroform-d)δ7.41(s,2H),7.36–7.20(m,6H),7.14–6.96(m,3H),4.92(s,2H),4.43(s,1H),3.70(s,3H),3.28(s,2H),3.18–2.92(m,2H),1.40(s,9H). 13 C NMR(100MHz,Chloroform-d)δ171.2,169.8,155.4,136.3,131.8,129.2,128.7,128.6,128.5,127.0,122.8,92.8,80.4,78.1,55.7,52.9,52.0,38.1,37.5,28.3.HRMS(ESI)m/z:calcd for C 26 H 30 N 2 O 5 SNa[M+Na] + :505.1768,found:505.1767.
example 2
(tert-Butoxycarbonyl) -L-phenylpropionyl-L-cysteine methyl ester 0.0764g (0.2 mmol,1.0 equiv.) was weighed out, dissolved in 2mL of acetonitrile, and 43. Mu.L of triethylamine (0.3 mmol,1.5 equiv.) was added as a first reaction solution. 0.1308g of an alkynyl reagent (0.3 mmol,1.5 equiv.) was weighed out and dissolved in 2mL of acetonitrile as a second reaction solution. The reaction solution was simultaneously pumped into a microreactor having an inner diameter of 0.6mm at a pumping flow rate of 0.15mL/min and a volume of 1.4mL, and the reaction residence time was 4.7min. After completion of the reaction, TLC was performed, the reaction solution was extracted with ethyl acetate and saturated brine (3X 25 mL), the organic layers were combined, dried over anhydrous sodium sulfate, and after the solvent was distilled off under reduced pressure, the final product was 81.9mg, yield 85% was obtained by silica gel column chromatography (petroleum ether: ethyl acetate=3:1).
Example 3
(tert-Butoxycarbonyl) -L-phenylpropionyl-L-cysteine methyl ester 0.0764g (0.2 mmol,1.0 equiv.) was weighed out and dissolved in 2mL of dimethyl sulfoxide, and 35. Mu.L of 2, 6-lutidine (0.3 mmol,1.5 equiv.) was added as a first reaction solution. 0.1308g of an alkynyl reagent (0.3 mmol,1.5 equiv.) was weighed out and dissolved in 2mL of dimethyl sulfoxide as a second reaction solution. The reaction solution was simultaneously pumped into a microreactor having an inner diameter of 0.6mm at a pumping flow rate of 0.15mL/min and a volume of 1.4mL, and the reaction residence time was 4.7min. After completion of the reaction, TLC was performed, the reaction solution was extracted with ethyl acetate and saturated brine (3X 25 mL), the organic layers were combined, dried over anhydrous sodium sulfate, and after the solvent was distilled off under reduced pressure, 80.0mg of the final product was obtained by silica gel column chromatography (petroleum ether: ethyl acetate=3:1), yield 83%.
Example 4
1.91g (5.0 mmol,1.0 equiv.) of methyl (t-butoxycarbonyl) -L-phenylpropionyl-L-cysteine was weighed out, dissolved in dimethyl sulfoxide, and 1041. Mu.L of triethylamine (7.5 mmol,1.5 equiv.) was added to prepare 10mL of a solution as a first reaction solution. 3.27g of an alkynyl reagent (7.5 mmol,1.5 equiv.) was weighed out and dissolved in dimethyl sulfoxide to prepare 10mL of a solution as a second reaction solution. The reaction solution was simultaneously pumped into a microreactor having an inner diameter of 0.6mm at a pumping flow rate of 0.15mL/min and a volume of 1.4mL, and the reaction residence time was 4.7min. After completion of the reaction, TLC was performed, the reaction solution was extracted with ethyl acetate and saturated brine (3X 100 mL), the organic layers were combined, dried over anhydrous sodium sulfate, and after the solvent was distilled off under reduced pressure, 2.12g of the final product was obtained by silica gel column chromatography (petroleum ether: ethyl acetate=3:1), in 88% yield.
Example 5
(tert-Butoxycarbonyl) -L-tryptophanyl-L-cysteine methyl ester 0.0842g (0.2 mmol,1.0 equiv.) was weighed out and dissolved in 2mL dimethyl sulfoxide, and 43. Mu.L triethylamine (0.3 mmol,1.5 equiv.) was added as a first reaction solution. 0.1308g of an alkynyl reagent (0.3 mmol,1.5 equiv.) was weighed out and dissolved in 2mL of dimethyl sulfoxide as a second reaction solution. The reaction solution was simultaneously pumped into a microreactor having an inner diameter of 0.6mm at a pumping flow rate of 0.15mL/min and a volume of 1.4mL, and the reaction residence time was 4.7min. After the reaction is completed, the reaction is carried outThe reaction mixture was extracted with ethyl acetate and saturated brine (3×25 mL), and the organic layers were combined, dried over anhydrous sodium sulfate, and after removal of the solvent by distillation under reduced pressure, the final product was obtained by silica gel column chromatography (petroleum ether: ethyl acetate=3:1) in a yield of 95%. Characterization data were as follows (fig. 5, fig. 6): 1 H NMR(400MHz,Chloroform-d)δ8.47(s,1H),7.59(d,J＝7.4Hz,1H),7.42–7.23(m,6H),7.20–7.13(m,1H),7.12–7.05(m,1H),7.04–6.87(m,2H),5.20(s,1H),4.83(s,1H),4.51(s,1H),3.61(s,3H),3.38–3.03(m,4H),1.42(s,9H). 13 C NMR(100MHz,Chloroform-d)δ171.9,169.8,155.5,136.3,131.7,128.5,128.4,127.5,123.3,122.9,122.2,119.7,118.7,111.4,110.1,92.9,80.3,78.1,52.8,51.8,37.6,31.6,28.3,22.7,14.2.HRMS(ESI)m/z:calcd for C 28 H 31 N 3 O 5 SNa[M+Na] + :544.1877,found:544.1880.
example 6
(tert-Butoxycarbonyl) -L-tyrosyl-L-cysteine methyl ester 0.0796g (0.2 mmol,1.0 equiv.) was weighed out and dissolved in 2mL dimethyl sulfoxide, and 43. Mu.L triethylamine (0.3 mmol,1.5 equiv.) was added as a first reaction solution. 0.1308g of an alkynyl reagent (0.3 mmol,1.5 equiv.) was weighed out and dissolved in 2mL of dimethyl sulfoxide as a second reaction solution. The reaction solution was simultaneously pumped into a microreactor having an inner diameter of 0.6mm at a pumping flow rate of 0.15mL/min and a volume of 1.4mL, and the reaction residence time was 4.7min. After completion of the reaction, TLC was performed, the reaction solution was extracted with ethyl acetate and saturated brine (3X 25 mL), the organic layers were combined, dried over anhydrous sodium sulfate, and after the solvent was distilled off under reduced pressure, the final product was obtained in a yield of 88% by silica gel column chromatography (petroleum ether: ethyl acetate=3:1). Characterization data were as follows (fig. 7, 8): 1 H NMR(400MHz,Chloroform-d)δ7.45–7.37(m,2H),7.34–7.25(m,3H),7.07(s,1H),6.99–6.84(m,3H),6.71(d,J＝7.6Hz,2H),5.14–4.99(m,1H),4.91(s,1H),4.37(s,1H),3.69(s,3H),3.31–3.19(m,2H),3.07–2.86(m,2H),1.42(s,9H). 13 C NMR(100MHz,Chloroform-d)δ171.7,169.9,155.6,155.3,131.7,130.3,128.6,128.5,127.5,122.8,115.7,93.0,80.7,77.9,55.9,53.0,52.0,37.4,31.6,28.3,22.7,14.2.HRMS(ESI)m/z:calcd for C 26 H 30 N 2 O 6 SNa[M+Na] + :521.1717,found:521.1709.
example 7
(tert-Butoxycarbonyl) -L-glutamyl-L-cysteine methyl ester 0.0726g (0.2 mmol,1.0 equiv.) was weighed out and dissolved in 2mL dimethyl sulfoxide, and 43. Mu.L triethylamine (0.3 mmol,1.5 equiv.) was added as the first reaction solution. 0.1308g of an alkynyl reagent (0.3 mmol,1.5 equiv.) was weighed out and dissolved in 2mL of dimethyl sulfoxide as a second reaction solution. The reaction solution was simultaneously pumped into a microreactor having an inner diameter of 0.6mm at a pumping flow rate of 0.15mL/min and a volume of 1.4mL, and the reaction residence time was 4.7min. After completion of the reaction, TLC was performed, the reaction solution was extracted with ethyl acetate and saturated brine (3X 25 mL), the organic layers were combined, dried over anhydrous sodium sulfate, and after the solvent was distilled off under reduced pressure, 77.8mg of the final product was obtained by silica gel column chromatography (petroleum ether: ethyl acetate=3:1), in 84% yield. Characterization data were as follows (fig. 9, fig. 10): 1 H NMR(400MHz,Chloroform-d)δ7.95(s,1H),7.47–7.37(m,2H),7.34–7.24(m,3H),6.55(s,1H),6.17(s,1H),5.74(s,1H),5.01–4.86(m,1H),4.34–4.22(m,1H),3.72(s,3H),3.38–3.18(m,2H),2.43-2.33(m,2H),2.11–1.96(m,2H),1.43(s,9H). 13 C NMR(100MHz,Chloroform-d)δ175.7,172.0,170.7,155.9,131.7,128.5,128.4,122.9,93.5,80.2,77.8,53.6,52.9,51.9,37.2,31.7,29.0,28.3.HRMS(ESI)m/z:calcd for C 22 H 29 N 3 O 6 SNa[M+Na] + :486.1669,found:486.1627.
example 8
Weighing (tert-butoxycarbonyl) -L-pentyl-L-prolyl0.0862g (0.2 mmol,1.0 equiv.) of L-cysteine methyl ester, dissolved in 2mL of dimethyl sulfoxide, was added 43. Mu.L of triethylamine (0.3 mmol,1.5 equiv.) as a first reaction solution. 0.1308g of an alkynyl reagent (0.3 mmol,1.5 equiv.) was weighed out and dissolved in 2mL of dimethyl sulfoxide as a second reaction solution. The reaction solution was simultaneously pumped into a microreactor having an inner diameter of 0.6mm at a pumping flow rate of 0.15mL/min and a volume of 1.4mL, and the reaction residence time was 4.7min. After completion of the reaction, TLC was performed, the reaction solution was extracted with ethyl acetate and saturated brine (3X 25 mL), the organic layers were combined, dried over anhydrous sodium sulfate, and after the solvent was distilled off under reduced pressure, 89.2mg of the final product was obtained by silica gel column chromatography (petroleum ether: ethyl acetate=3:1), in 84% yield. Characterization data were as follows (fig. 11, fig. 12): 1 H NMR(400MHz,Chloroform-d)δ7.60(s,1H),7.43–7.37(m,2H),7.32–7.26(m,3H),5.30(s,1H),4.91–4.83(m,1H),4.70–4.60(m,1H),4.37–4.24(m,1H),3.75–3.67(m,4H),3.62–3.56(m,1H),3.36–3.22(m,2H),2.34(d,J＝9.6Hz,1H),2.10–1.93(m,4H),1.43(s,9H),1.03(d,J＝6.7Hz,3H),0.96(d,J＝6.6Hz,3H). 13 C NMR(100MHz,Chloroform-d)δ172.7,171.1,170.1,155.9,131.6,128.4,128.3,123.0,93.1,79.6,77.9,60.0,56.8,52.8,51.9,47.6,37.5,31.6,28.3,27.4,25.1,19.7,17.4.HRMS(ESI)m/z:calcd for C 27 H 37 N 3 O 6 SNa[M+Na] + :554.2295,found:554.2247.
example 9
(tert-Butoxycarbonyl) -L-phenylpropionyl-L-cysteine methyl ester 0.0764g (0.2 mmol,1.0 equiv.) was weighed out and dissolved in 2mL of dimethyl sulfoxide, and 43. Mu.L of triethylamine (0.3 mmol,1.5 equiv.) was added as a first reaction solution. 0.1512g of an alkynyl reagent (0.3 mmol,1.5 equiv.) was weighed out and dissolved in 2mL of dimethyl sulfoxide as a second reaction solution. The reaction solution was simultaneously pumped into a microreactor having an inner diameter of 0.6mm at a pumping flow rate of 0.15mL/min and a volume of 1.4mL, and the reaction residence time was 4.7min. After completion of the reaction, TLC was performed, and the reaction solution was extracted with ethyl acetate and saturated brine (3X 25 mL), the organic layers were combined, dried over anhydrous sodium sulfate, and the solvent was removed by distillation under the reduced pressure, followed by column chromatography over silica gel (petroleum ether: ethyl acetate=3:1) to give 93.5mg of the final product in 85% yield. Characterization data were as follows (fig. 13, 14, 15): 1 H NMR(400MHz,Chloroform-d)δ7.55(d,J＝8.3Hz,2H),7.49(d,J＝8.1Hz,2H),7.30–7.22(m,3H),7.13(d,J＝6.9Hz,2H),7.02(s,1H),4.95(d,J＝25.7Hz,2H),4.44(s,1H),3.71(s,3H),3.30(d,J＝4.7Hz,2H),3.16–3.00(m,2H),1.40(s,9H). 13 C NMR(100MHz,Chloroform-d)δ171.3,169.8,155.4,136.3,131.5,129.9(d,J＝32.6Hz,1C),129.2,128.7,127.0,126.7,125.3(8)(d,J＝38.5Hz,1),125.3(7)(q,J＝3.8Hz,2C),122.5,91.8,81.5,80.4,55.7,52.7,51.9,38.1,37.5,28.2. 19 F NMR(376MHz,Chloroform-d)δ62.82.HRMS(ESI)m/z:calcd for C 27 H 29 F 3 N 2 O 5 SNa[M+Na] + :573.1641,found:573.1619.
example 10
(tert-Butoxycarbonyl) -L-phenylpropionyl-L-cysteine methyl ester 0.0764g (0.2 mmol,1.0 equiv.) was weighed out and dissolved in 2mL of dimethyl sulfoxide, and 43. Mu.L of triethylamine (0.3 mmol,1.5 equiv.) was added as a first reaction solution. 0.1476g of an alkynyl reagent (0.3 mmol,1.5 equiv.) was weighed out and dissolved in 2mL of dimethyl sulfoxide as a second reaction solution. The reaction solution was simultaneously pumped into a microreactor having an inner diameter of 0.6mm at a pumping flow rate of 0.15mL/min and a volume of 1.4mL, and the reaction residence time was 4.7min. After completion of the reaction, TLC was performed, the reaction solution was extracted with ethyl acetate and saturated brine (3X 25 mL), the organic layers were combined, dried over anhydrous sodium sulfate, and after the solvent was distilled off under reduced pressure, 89.3mg of the final product was obtained by silica gel column chromatography (petroleum ether: ethyl acetate=3:1), yield 83%. Characterization data were as follows (fig. 16, fig. 17): 1 H NMR(400MHz,Chloroform-d)δ7.39–7.30(m,4H),7.28–7.20(m,3H),7.13–7.02(m,3H),4.92(s,2H),4.43(s,1H),3.71(s,3H),3.27(d,J＝4.3Hz,2H),3.20–3.12(m,1H),3.01–2.88(m,1H),1.40(s,9H),1.30(s,9H). 13 C NMR(100MHz,Chloroform-d)δ171.2,169.9,155.3,152.0,136.5,131.7,129.2,128.7,127.0,125.5,119.8,93.3,80.4,55.7,52.9,52.1,38.1,37.5,34.8,31.2,28.2.HRMS(ESI)m/z:calcd for C 30 H 39 N 2 O 5 SNa[M+Na] + :561.2394,found:561.2360.
example 11
(tert-Butoxycarbonyl) -L-phenylpropionyl-L-cysteine methyl ester 0.0764g (0.2 mmol,1.0 equiv.) was weighed out and dissolved in 2mL of dimethyl sulfoxide, and 43. Mu.L of triethylamine (0.3 mmol,1.5 equiv.) was added as a first reaction solution. 0.1548g of an alkynyl reagent (0.3 mmol,1.5 equiv.) was weighed out and dissolved in 2mL of dimethyl sulfoxide as a second reaction solution. The reaction solution was simultaneously pumped into a microreactor having an inner diameter of 0.6mm at a pumping flow rate of 0.15mL/min and a volume of 1.4mL, and the reaction residence time was 4.7min. After completion of the reaction, TLC was performed, the reaction solution was extracted with ethyl acetate and saturated brine (3X 25 mL), the organic layers were combined, dried over anhydrous sodium sulfate, and after the solvent was distilled off under reduced pressure, 96.7mg of the final product was obtained by silica gel column chromatography (petroleum ether: ethyl acetate=3:1), in 86% yield. Characterization data were as follows (fig. 18, fig. 19): 1 H NMR(400MHz,Chloroform-d)δ7.30(t,J＝7.2Hz,2H),7.25(d,J＝7.1Hz,1H),7.20(d,J＝7.0Hz,2H),6.76(s,1H),5.02(s,1H),4.84–4.76(m,1H),4.48–4.32(m,1H),3.75(s,3H),3.28–3.11(m,1H),3.15–3.06(m,3H),1.41(s,9H),1.07(s,21H). 13 C NMR(100MHz,Chloroform-d)δ171.2,169.9,155.3,136.3,129.3,128.7,127.0,98.2,94.1,80.3,55.5,52.8,51.7,38.1,28.3,18.6,11.3.HRMS(ESI)m/z:calcd for C 29 H 46 SiN 2 O 5 SNa[M+Na] + :585.2789,found:585.2757.
example 12
Weighing (t-butyl)Oxycarbonyl) -L-phenylpropionyl-L-cysteine methyl ester 0.0764g (0.2 mmol,1.0 equiv.) was dissolved in 2mL dimethyl sulfoxide, and 43 μl triethylamine (0.3 mmol,1.5 equiv.) was added as a first reaction solution. 0.1458g of an alkynyl reagent (0.3 mmol,1.5 equiv.) was weighed out and dissolved in 2mL of dimethyl sulfoxide as a second reaction solution. The reaction solution was simultaneously pumped into a microreactor having an inner diameter of 0.6mm at a pumping flow rate of 0.15mL/min and a volume of 1.4mL, and the reaction residence time was 4.7min. After completion of the reaction, TLC was performed, the reaction solution was extracted with ethyl acetate and saturated brine (3X 25 mL), the organic layers were combined, dried over anhydrous sodium sulfate, and after the solvent was distilled off under reduced pressure, 88.3mg of the final product was obtained by silica gel column chromatography (petroleum ether: ethyl acetate=3:1), yield 83%. Characterization data were as follows (fig. 20, fig. 21): 1 H NMR(400MHz,Chloroform-d)δ8.27(d,J＝8.2Hz,1H),7.83(t,J＝8.4Hz,2H),7.66(d,J＝6.8Hz,1H),7.62–7.56(m,1H),7.55–7.49(m,1H),7.40(t,J＝7.7Hz,1H),7.22(q,J＝11.0,10.5Hz,3H),7.07(s,1H),7.01(d,J＝6.6Hz,2H),4.93(d,J＝47.6Hz,2H),4.42(s,1H),3.65(s,3H),3.38(d,J＝3.7Hz,2H),3.16–3.07(m,1H),2.96–2.83(m,1H),1.39(s,9H). 13 C NMR(100MHz,Chloroform-d)δ171.2,169.8,155.4,136.3,133.4,133.2,130.9,129.2,129.1,128.7,128.4,127.1,127.0,126.6,126.1,125.3,120.5,91.0,82.9,55.7,52.9,52.2,37.9,28.2.HRMS(ESI)m/z:calcd for C 30 H 32 N 2 O 5 SNa[M+Na] + :555.1924,found:555.1915.
example 13
Methyl N-acetyl-L-cysteinate (0.0354 g) (0.2 mmol,1.0 equiv.) was weighed out and dissolved in 2mL dimethyl sulfoxide, and 43. Mu.L triethylamine (0.3 mmol,1.5 equiv.) was added as a first reaction solution. 0.1308g of an alkynyl reagent (0.3 mmol,1.5 equiv.) was weighed out and dissolved in 2mL of dimethyl sulfoxide as a second reaction solution. The reaction solution was simultaneously pumped into a microreactor having an inner diameter of 0.6mm at a pumping flow rate of 0.15mL/min and a volume of 1.4mL, and the reaction residence time was 4.7min. TLC detection after the reaction was completed, the reaction was reversedThe reaction mixture was extracted with ethyl acetate and saturated brine (3×25 mL), and the organic layers were combined, dried over anhydrous sodium sulfate, and after the solvent was removed by distillation under the reduced pressure, the resultant was subjected to silica gel column chromatography (petroleum ether: ethyl acetate=3:1) to give 48.2mg of the final product in a yield of 87%. Characterization data were as follows (fig. 22, fig. 23): 1 H NMR(400MHz,Chloroform-d)δ7.45–7.37(m,2H),7.36–7.28(m,3H),6.58(s,1H),5.05–4.95(m,1H),3.74(s,3H),3.33(d,J＝4.3Hz,2H),2.04(s,3H). 13 C NMR(100MHz,Chloroform-d)δ170.4,169.9,131.6,128.6,128.4,122.8,92.8,78.1,53.0,52.2,37.5,23.2.HRMS(ESI)m/z:calcd for C 14 H 15 NO 3 SNa[M+Na] + :300.0665,found:300.0675.
comparative example 1
Methyl (t-butoxycarbonyl) -L-phenylpropionyl-L-cysteinate 0.0764g (0.2 mmol,1.0 equiv.) was weighed out, 0.1308g of alkynyl reagent (0.3 mmol,1.5 equiv.) was added to a Schlenk reaction tube, replaced with argon three times, and then added to 2mL of dimethyl sulfoxide and 43. Mu.L of triethylamine (0.3 mmol,1.5 equiv.). After completion of the reaction at room temperature, TLC was performed, the reaction solution was extracted with ethyl acetate and saturated brine (3X 25 mL), the organic layers were combined, dried over anhydrous sodium sulfate, and after removal of the solvent by distillation under reduced pressure, the final product was 79.0mg, yield 82% was obtained by silica gel column chromatography (petroleum ether: ethyl acetate=3:1).
The invention provides a thought and a method for realizing alkynyl functional modification of cysteine and polypeptide thereof by utilizing a microchannel reactor, and the method and the way for realizing the technical scheme are numerous, the above is only a preferred embodiment of the invention, and it should be pointed out that a plurality of improvements and modifications can be made by those skilled in the art without departing from the principle of the invention, and the improvements and the modifications are also considered as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.
Claims (8)
1. A method for performing alkynyl functionalization of cysteine-containing polypeptides using a microchannel reactor, comprising the steps of:
(1) Dissolving a polypeptide containing cysteine shown in a formula I in a first solvent to obtain a first reaction solution; the first reaction liquid also comprises organic alkali;
(2) Dissolving an alkynyl functionalization reagent shown in a formula III in a second solvent to obtain a second reaction solution;
(3) Pumping the first reaction liquid and the second reaction liquid into a micro-channel reactor respectively and simultaneously for reaction, and collecting effluent liquid to obtain a reaction liquid containing an alkynyl functionalized product of the polypeptide containing cysteine shown in the formula II;
wherein, the liquid crystal display device comprises a liquid crystal display device,
R 1 is methyl;
R 2 any one selected from structures shown in a formula IV;
R 3 selected from phenyl, 4-trifluoromethylphenyl, 4-tert-butylphenyl, triisopropylsilyl, or naphthalene;
the concentration of the polypeptide containing cysteine in the first reaction solution is 0.05-1.0 mmol/mL; the molar ratio of cysteine-containing polypeptide to organic base is 1: 1-5;
in the second reaction solution, the concentration of the alkynyl functional reagent is 0.05-2.0 mmol/mL.
2. The method according to claim 1, wherein the concentration of the cysteine-containing polypeptide in the first reaction solution is 0.05 to 0.5mmol/mL.
3. The method according to claim 1, wherein the molar ratio of cysteine-containing polypeptide to organic base in the first reaction solution is 1:1.5.
4. the method of claim 1, wherein the concentration of the alkynyl functionalizing agent in the second reaction solution is 0.15 to 1.0mmol/mL.
5. The method of claim 1, wherein the first solvent and the second solvent are each independently selected from methanol, ethanol, acetone, 1, 4-dioxane, methylene chloride, 1, 2-dichloroethane, N-dimethyl propenyl urea, acetonitrile, N-dimethylformamide, N-dimethylacetamide, water, phosphate buffer, tetrahydrofuran, dimethylsulfoxide, or any combination thereof.
6. The method of claim 1, wherein the first and second reaction fluids are pumped at a rate of 1:1.
7. the method of claim 1, wherein the temperature of the reaction is room temperature.
8. The method of claim 1, wherein the reaction time is 30s to 2.6 hours.
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