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,,
R1 selected from hydrogen, methyl, ethyl, propyl or isopropyl, preferably methyl;
R2 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:
R3 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 is3 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%.
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 C26 H30 N2 O5 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 C28 H31 N3 O5 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 C26 H30 N2 O6 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 C22 H29 N3 O6 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 C27 H37 N3 O6 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 C27 H29 F3 N2 O5 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 C30 H39 N2 O5 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 C29 H46 SiN2 O5 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 C30 H32 N2 O5 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 C14 H15 NO3 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.