CN113896762B - Liquid phase synthesis method of biotin tripeptide-1 - Google Patents
Liquid phase synthesis method of biotin tripeptide-1 Download PDFInfo
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Abstract
The invention discloses a liquid phase synthesis method of biotin tripeptide-1, relating to the technical field of polypeptide synthesis and comprising the following steps: using functional ionic liquid as a carrier, bonding Boc-Lys-OH through condensation reaction, connecting with Trt-His-OH through dehydration condensation, and then cutting off the functional ionic liquid in an alkaline environment to obtain His (Trt) -Lys (Boc) -OH; then the Biotin tripeptide-1 is obtained by chemical reaction and glycine bonding with Biotin-N-hydroxysuccinimide ester to obtain Biotin-Gly-OH through dehydration condensation reaction and deprotection. The purity and yield of the biotin tripeptide-1 prepared by the liquid phase synthesis method are obviously increased; and the intermediate product is simple to separate and purify, the cost is reduced, the pollution is reduced, and the expanded production can be realized.
Description
Technical Field
The invention belongs to the technical field of polypeptide synthesis, and particularly relates to a liquid-phase synthesis method of biotin tripeptide-1.
Background
In the prior art, biotin tripeptide-1 is synthesized by a solid phase method, and has the main problems that intermediate heteropeptide on a solid phase carrier cannot be separated and is purified by preparing a liquid phase finally, so that the product is more impure and difficult to purify due to amplification, and the problems of high cost, large pollution to the environment and small single-batch secondary yield exist.
As a green solvent and a catalyst emerging in recent years, the ionic liquid has obvious advantages in a plurality of important organic synthesis reactions: the selectivity, speed and yield of the reaction are obviously improved, the post-treatment of the reaction is convenient, the ionic liquid can be recycled, and the like. The liquid phase parallel synthesis method using the functionalized ionic liquid as the carrier integrates the advantages of liquid phase synthesis and solid phase synthesis, so that the advantages in the aspect of liquid phase parallel synthesis of small molecules are more and more obvious. Ionic liquid is used as a carrier to load organic unit reaction and synthesis of small molecular compounds, and a method for synthesizing heterocyclic compounds, polypeptides, oligosaccharides and other compounds is established; however, almost no report has been made on the method for producing biotin tripeptide-1.
Disclosure of Invention
The invention aims to provide a liquid phase synthesis method of biotin tripeptide-1, wherein the purity and yield of the biotin tripeptide-1 prepared by the liquid phase synthesis method are obviously improved; and the intermediate product is simple to separate and purify, the cost is reduced, the pollution is reduced, and the expanded production can be realized.
The technical scheme adopted by the invention for realizing the purpose is as follows:
a liquid phase synthesis method of biotin tripeptide-1 comprises the following steps:
preparation of His (Trt) -Lys (Boc) -OH:
-bonding Boc-Lys-OH by condensation reaction using functionalized ionic liquid as carrier to obtain an ionophore A;
connecting the ionophore A with Trt-His-OH through dehydration condensation to obtain an ionophore B;
cutting the ionophore B in an alkaline solution environment to remove the functionalized ionic liquid to obtain His (Trt) -Lys (Boc) -OH;
preparation of Biotin-Gly-OH:
-Biotin-Gly-OH is obtained by chemical reaction of Biotin-N-hydroxysuccinimide ester with glycine;
preparation of biotin tripeptide-1:
his (Trt) -Lys (Boc) -OH and Biotin-Gly-OH are subjected to dehydration condensation reaction and deprotection to obtain Biotin tripeptide-1;
wherein the functionalized ionic liquid structure at least comprises a hydroxyl active group; the raw material of the functionalized ionic liquid at least comprises 2-chloro-6, 7-dimethoxy-3H-quinazoline-4-ketone or 4-chloro-1-butanol. The invention establishes a method for synthesizing biotin tripeptide-1 by taking functional ionic liquid as a carrier, carrying out a cutting reaction by using an alkaline solution under a mild condition and carrying out a liquid phase synthesis. The method adopts the functionalized ionic liquid for liquid phase synthesis, and has the advantages of high unit load and simple and convenient separation and purification of intermediate products; the use amounts of organic volatile solvent, excessive reagent, catalyst and the like used in the reaction process are reduced, and the pollution to the environment is reduced; and the synthesized target product has high yield and purity, and does not need further chromatographic purification. The invention adds the functionalized ionic liquid prepared by 2-chloro-6, 7-dimethoxy-3H-quinazoline-4-ketone as a soluble carrier to be used in the liquid phase synthesis of biotin tripeptide-1, can further improve the purity of the biotin tripeptide-1 product, and obviously increases the yield.
In some embodiments, the functionalized ionic liquid feedstock composition further comprises 1-methylimidazole.
Further, a liquid phase synthesis method of biotin tripeptide-1 comprises the following steps:
preparation of His (Trt) -Lys (Boc) -OH:
mixing, stirring and dissolving a functionalized ionic liquid, Boc-Lys-OH, DMAP and acetonitrile, adding a DCC/dichloromethane solution with the concentration of 0.8-1M, stirring and reacting for 24-28 h under the conditions of room temperature and nitrogen protection, performing suction filtration by using a Buchner funnel paved with two layers of filter paper and diatomite, performing reduced pressure distillation and ether washing, dissolving in dichloromethane, washing with hydrochloric acid with the concentration of 1.8-2M, adding anhydrous sodium sulfate into an organic phase, drying, performing reduced pressure distillation and concentration, and performing vacuum drying at 40-50 ℃ for 24-30 h to obtain an ionophore A;
mixing an ionophore A, PyBOP, Trt-His-OH, DIPEA and acetonitrile, stirring and dissolving under the protection of nitrogen, stirring and reacting for 8-10 h under the condition of constant-temperature water bath at 35-40 ℃, distilling under reduced pressure at 50-55 ℃ to remove a solvent, sequentially washing with diethyl ether and deionized water for 3-4 times respectively, adding anhydrous sodium sulfate, drying, and vacuum drying for 24-30 h to obtain an ionophore B;
taking 0.8-1M sodium hydroxide, THF/water (v/v, 1: 1.5-2.5) and an ionophore B, stirring at room temperature under the protection of nitrogen, carrying out TLC monitoring reaction, carrying out reduced pressure distillation at 50-55 ℃, adding 1.8-2M hydrochloric acid to acidify until the pH is 5-6, filtering, washing precipitates for 2-3 times with deionized water, and carrying out vacuum drying at 70-80 ℃ for 24-30 h to obtain His (Trt) -Lys (Boc) -OH;
preparation of Biotin-Gly-OH:
mixing Biotin-N-hydroxysuccinimide ester and glycine, adding a tetrahydrofuran/water mixed solution (v/v, 1: 0.8-1.4) for dissolving, adding a sodium hydroxide solution with the concentration of 25-30 wt% for adjusting the pH value to 8-9, reacting at room temperature, monitoring the reaction by TLC, adjusting the pH value to 2.0-2.5 after the reaction is finished, separating out a solid, filtering, leaching with water, and drying in vacuum to obtain Biotin-Gly-OH;
preparation of biotin tripeptide-1:
adding N, N' -carbonyl imidazole into Biotin-Gly-OH and tetrahydrofuran under an ice bath cooling condition, dropwise adding the reaction liquid into a tetrahydrofuran solution containing His (Trt) -Lys (Boc) -OH and triethylamine in a concentration of 8-10 wt% when the reaction liquid does not bubble any more, reacting at room temperature after finishing dropping, monitoring the reaction by TLC, concentrating under reduced pressure after finishing the reaction, adding water and ethyl acetate, adjusting the pH to 2-2.5 by using hydrochloric acid with a concentration of 1.8-2M, washing an organic phase by using brine, drying by using anhydrous sodium sulfate, and concentrating under reduced pressure to obtain a product M; and (2) deprotection, namely adding dichloromethane into the product M under the protection of nitrogen to dissolve the product M, adding isovolumetric TFA, stirring at room temperature for 0.5-1 h, monitoring the reaction by TLC, evaporating the solvent at 50-55 ℃ under reduced pressure after the reaction is finished, washing with ethyl ether for 2-4 times, and drying in vacuum at 40-50 ℃ for 24-30 h to obtain a product N, namely the biotin tripeptide-1.
In some embodiments, the molar ratio of the functionalized ionic liquid to the Boc-Lys-OH during the preparation of His (Trt) -Lys (Boc) -OH is 1: 1.8-2.5; the molar ratio of the functionalized ionic liquid to the DMAP is 1: 0.3-0.5; the solid-to-liquid ratio of the functionalized ionic liquid to the acetonitrile is 1 g: 24-28 mL; the molar ratio of the functionalized ionic liquid to the DCC is 1: 2-2.4.
In some embodiments, the molar ratio of the ionophore A, Trt-His-OH during the preparation of His (Trt) -Lys (Boc) -OH is 1:1.4 to 1.6; the molar ratio of the ionophore A, PyBOP is 1: 1.4-1.6; the molar ratio of the ionophore A, DIPEA is 1: 2.5-3.5; the solid-liquid ratio of the ionophore A to the acetonitrile is 1 g: 22-26 mL.
In some embodiments, the volume ratio of the sodium hydroxide to the THF/water in the preparation of His (Trt) -Lys (Boc) -OH is 1: 14-16; the solid-to-liquid ratio of the ionophore B to the sodium hydroxide is 1 g: 0.8-1 mL.
In some embodiments, the Biotin-Gly-OH is prepared in a molar ratio of Biotin-N-hydroxysuccinimide ester to glycine of 1: 1.1-1.3; the solid-to-liquid ratio of the biotin-N-hydroxysuccinimide ester to the tetrahydrofuran/water mixed solution is 1 g: 12-15 mL.
In some embodiments, during the preparation process of the Biotin tripeptide-1, the solid-to-liquid ratio of Biotin-Gly-OH and tetrahydrofuran is 1 g: 8-12 mL; the mass ratio of Biotin-Gly-OH to N, N' -carbonyl imidazole is 1: 0.5-0.6; the mass ratio of His (Trt) -Lys (Boc) -OH to Biotin-Gly-OH is 1: 1.5-2; the mass ratio of triethylamine to Biotin-Gly-OH is 1: 0.35-0.45.
In some embodiments, biotin tripeptide-1 is greater than or equal to 96% pure with > 90% yield; preferably, the purity of the biotin tripeptide-1 is more than or equal to 97 percent, and the yield is more than 94 percent; more preferably, the biotin tripeptide-1 is > 99% pure.
The preparation method of the functionalized ionic liquid comprises the following steps:
mixing 1-methylimidazole, 4-chloro-1-butanol or 2-chloro-6, 7-dimethoxy-3H-quinazoline-4-ketone, heating to 80-90 ℃, and reacting in a nitrogen atmosphere to obtain the compound.
Specifically, the preparation method of the functionalized ionic liquid comprises the following steps:
mixing 1-methylimidazole and 4-chloro-1-butanol, heating to 80-90 ℃, and reacting for 48-54 h in a nitrogen atmosphere; then adding equal volume of ether to wash for 4-6 times, violently stirring, and carrying out vacuum drying at 50-60 ℃ overnight to obtain a functionalized ionic liquid;
or the like, or, alternatively,
mixing 1-methylimidazole and 2-chloro-6, 7-dimethoxy-3H-quinazoline-4-ketone, heating to 80-90 ℃, and reacting for 48-54H in a nitrogen atmosphere; and then adding equal volume of diethyl ether to wash for 4-6 times, violently stirring, and carrying out vacuum drying at 50-60 ℃ overnight to obtain the functionalized ionic liquid.
In some embodiments, the molar ratio of 1-methylimidazole to 4-chloro-1-butanol is 1:1.2 to 1.6; or the molar ratio of the 1-methylimidazole to the 2-chloro-6, 7-dimethoxy-3H-quinazoline-4-one is 1: 1.2-1.6.
Preferably, further purification is performed during the preparation of His (Trt) -Lys (Boc) -OH, and/or during the preparation of biotin tripeptide-1 using functionalized macroporous resins; wherein, the raw materials of the functional macroporous resin comprise macroporous absorption resin of poly (GMA-co-EDMA) matrix, 4-amino-4-deoxy-10-methylpteroic acid, m-phenylenediamine and m-phthalaldehyde. According to the invention, the macroporous adsorption resin of the poly (GMA-co-EDMA) matrix is modified by adopting 4-amino-4-deoxy-10-methylpteroic acid to obtain the functionalized macroporous resin, so that the adsorption performance of the resin is obviously improved, and the adsorption equivalent and desorption rate are effectively increased; the use stability of the macroporous resin is also enhanced, and after the macroporous resin is recycled for many times, the macroporous resin still keeps higher adsorption-desorption performance, has excellent regeneration and recycling performance, and effectively prolongs the service life of the macroporous resin; meanwhile, the modified macroporous resin has excellent selective adsorption on the biotin tripeptide-1. The method is applied to the liquid phase synthesis of the biotin tripeptide-1, and the purity and yield of the prepared product are obviously improved; and the catalyst is used in different stages of liquid phase synthesis, and has excellent influence; the intermediate-stage product is separated and purified, so that the purity and yield of the biotin tripeptide-1 product can be further improved; the final product is separated and purified, so that the purity of the product can be further improved.
Further, in the preparation process of His (Trt) -Lys (Boc) -OH, the obtained ionophore B is dissolved in 50-60% ethanol solution, and the concentration is 0.02-0.05 g/mL; adding functional macroporous resin for adsorption-desorption, separation and purification, reduced pressure distillation, vacuum drying, and then carrying out the ionic liquid carrier cutting step.
Further, in the preparation process of the biotin tripeptide-1, the obtained product N is dissolved in 50-60% ethanol solution, and the concentration is 0.02-0.05 g/mL; adding functional macroporous resin for adsorption-desorption, separation and purification, reduced pressure distillation and vacuum drying to obtain the biotin tripeptide-1.
In some embodiments, the solid-to-liquid ratio of the functionalized macroporous resin to the ethanol solution is 1 g: 50-60 mL.
Furthermore, the functionalized macroporous resin is obtained by mixing the raw material components and modifying the macroporous adsorption resin of the poly (GMA-co-EDMA) matrix by adopting a one-pot method through a physical and chemical reaction.
Specifically, the preparation method of the functionalized macroporous resin comprises the following steps:
mixing and dispersing macroporous adsorption resin of poly (GMA-co-EDMA) matrix, 4-amino-4-deoxy-10-methylpteroic acid, M-phenylenediamine and M-phthalaldehyde in dioxane, adding an acetic acid aqueous solution with the concentration of 2-4M, placing the system in an oil bath condition at the temperature of 80-90 ℃, and stirring and reacting for 24 hours in a nitrogen atmosphere; and centrifuging, collecting the precipitate, washing with THF and methanol for 3-5 times in sequence, and vacuum drying at 110-120 ℃ overnight to obtain the functionalized macroporous resin.
In some embodiments, the mass ratio of the macroporous adsorbent resin of the poly (GMA-co-EDMA) matrix, the 4-amino-4-deoxy-10-methylpteroic acid, the m-phenylenediamine, and the m-phthalaldehyde is 1: 0.15-0.3: 0.1-0.2: 0.1-0.25; the solid-to-liquid ratio of macroporous adsorption resin of a poly (GMA-co-EDMA) matrix to dioxane is 14-18 mg/mL; the volume of the acetic acid aqueous solution is 2-4% of that of dioxane.
The invention also discloses application of the functionalized ionic liquid as a carrier material and a catalyst.
The invention also discloses application of the functionalized ionic liquid in the field of polypeptide liquid phase synthesis.
In some embodiments, the use of a functionalized ionic liquid to increase the purity and yield of a polypeptide product.
Compared with the prior art, the invention has the following beneficial effects:
the invention adds the functionalized ionic liquid prepared by 2-chloro-6, 7-dimethoxy-3H-quinazoline-4-ketone as a soluble carrier to be used in the liquid phase synthesis of biotin tripeptide-1, can further improve the purity of the biotin tripeptide-1 product, and obviously increases the yield. Meanwhile, the method adopts 4-amino-4-deoxy-10-methylpteroic acid to modify the macroporous adsorption resin of the poly (GMA-co-EDMA) matrix to obtain the functionalized macroporous resin, and the adsorption equivalent and desorption rate of the functionalized macroporous resin are effectively increased; and has excellent regeneration and recycling performance; has excellent selective adsorption to the biotin tripeptide-1; the method is applied to the liquid phase synthesis of the biotin tripeptide-1, and the purity and the yield of the prepared product are further improved.
Therefore, the invention provides a liquid phase synthesis method of biotin tripeptide-1, and the purity and yield of the biotin tripeptide-1 prepared by the liquid phase synthesis method are obviously improved; and the intermediate product is simple to separate and purify, the cost is reduced, the pollution is reduced, and the expanded production can be realized.
Drawings
FIG. 1 shows the IR spectrum test results of the functionalized ionic liquid in test example 1 of the present invention;
FIG. 2 shows the IR spectrum of the functionalized macroporous resin of test example 1.
Detailed Description
The technical solution of the present invention is further described in detail below with reference to the following detailed description and the accompanying drawings:
the preparation of macroporous adsorption resin with a poly (GMA-co-EDMA) matrix in the embodiment of the invention is the prior art, and the basic preparation process is as follows:
adding styrene, PVP (the mass ratio of the styrene to the PVP is 1: 0.2) and AIBN (the addition amount is 1 wt% of the styrene) into absolute ethyl alcohol (the mass ratio of the styrene to the absolute ethyl alcohol is 1: 8) together, and performing ultrasonic dispersion for 30 min; then under the condition of oil bath at 80 ℃, nitrogen is filled for 15 min, and the reaction is carried out for 24 h in a sealed reflux manner; cooling to room temperature, centrifuging, removing the upper layer, adding water/industrial alcohol mixed solvent (w/w, 1: 1), ultrasonically dispersing, centrifuging, precipitating for 5 times, and vacuum drying for 24 h to obtain polystyrene microsphere seeds;
dispersing polystyrene microsphere seeds into an aqueous solution containing 1 wt% of PVA and 0.25 wt% of SDS, wherein the concentration is 20 mg/mL, and obtaining a solution A; dissolving GMA, EDMA (molar ratio of 1: 1) and AIBN (addition amount is 1.1 wt% of total amount of GMA and EDMA) in acetone solution (mass ratio of GMA to acetone is 1: 8) to obtain solution B; mixing the solution A and the solution B (the mass ratio is 1.5: 1), performing ultrasonic emulsification, swelling at 30 ℃ for 12 hours, and then placing the system at 70 ℃ for constant-temperature reaction for 24 hours; washing the obtained product with THF and methanol for 4 times, and vacuum drying at 60 deg.C for 24 hr to obtain macroporous adsorbent resin with poly (GMA-co-EDMA) matrix.
Example 1:
preparing a functionalized ionic liquid:
mixing 1-methylimidazole and 2-chloro-6, 7-dimethoxy-3H-quinazoline-4-ketone according to the molar ratio of 1:1.45, heating to 86 ℃, and reacting for 50 hours in a nitrogen atmosphere; then adding equal volume of ether to wash for 5 times, stirring vigorously, and drying overnight under vacuum at 55 ℃ to obtain the functionalized ionic liquid.
A liquid phase synthesis method of biotin tripeptide-1 comprises the following steps:
preparation of His (Trt) -Lys (Boc) -OH:
mixing, stirring and dissolving a functionalized ionic liquid, Boc-Lys-OH, DMAP and acetonitrile, adding a DCC/dichloromethane solution with the concentration of 1M, stirring and reacting for 25 hours at room temperature under the protection of nitrogen, performing suction filtration by using a Buchner funnel paved with two layers of filter paper and diatomite, performing reduced pressure distillation, washing with diethyl ether, dissolving in dichloromethane, washing with hydrochloric acid with the concentration of 2M, adding anhydrous sodium sulfate into an organic phase, drying, performing reduced pressure distillation and concentration, and performing vacuum drying at 45 ℃ for 24 hours to obtain an ionophore A; wherein the mole ratio of the functionalized ionic liquid to Boc-Lys-OH is 1: 2.2; the molar ratio of the functionalized ionic liquid to the DMAP is 1: 0.42; the solid-to-liquid ratio of the functionalized ionic liquid to the acetonitrile is 1 g:26.2 mL; the molar ratio of the functionalized ionic liquid to the DCC is 1: 2.2;
mixing an ionophore A, PyBOP, Trt-His-OH, DIPEA and acetonitrile, stirring and dissolving under the protection of nitrogen, stirring and reacting for 10 hours under the condition of constant-temperature water bath at 38 ℃, distilling under reduced pressure at 55 ℃ to remove a solvent, sequentially washing for 4 times by using diethyl ether and deionized water, adding anhydrous sodium sulfate, drying, and vacuum-drying for 24 hours to obtain an ionophore B; wherein the mol ratio of the ionophore A, Trt-His-OH is 1: 1.52; the molar ratio of the ionophore A, PyBOP was 1: 1.51; the molar ratio of the ionophore A, DIPEA was 1: 3.05; the solid-to-liquid ratio of the ionophore A to the acetonitrile is 1 g:24 mL;
taking 1M sodium hydroxide, THF/water (v/v, 1: 2.1) and an ionophore B, stirring at room temperature under the protection of nitrogen, monitoring the reaction by TLC, distilling at 55 ℃ under reduced pressure, adding 2M hydrochloric acid to acidify until the pH is 5.6, filtering, washing precipitates with deionized water for 3 times, and drying in vacuum at 76 ℃ for 30 hours to obtain (His Trt) -Lys (Boc) -OH; wherein the volume ratio of the sodium hydroxide to the THF to the water is 1: 15.5; the solid-to-liquid ratio of the ionophore B to the sodium hydroxide is 1 g:0.92 mL;
preparation of Biotin-Gly-OH:
mixing Biotin-N-hydroxysuccinimide ester and glycine, adding a tetrahydrofuran/water mixed solution (v/v, 1: 1.15) for dissolving, adding a sodium hydroxide solution with the concentration of 27.8 wt% for regulating the pH value to 8.5, reacting at room temperature, monitoring the reaction by TLC, regulating the pH value to 2.0 after the reaction is finished, separating out a solid, filtering, leaching with water, and drying in vacuum to obtain Biotin-Gly-OH; wherein the molar ratio of the biotin-N-hydroxysuccinimide ester to the glycine is 1: 1.22; the solid-to-liquid ratio of the biotin-N-hydroxysuccinimide ester to the tetrahydrofuran/water mixed solution is 1 g:13.5 mL;
preparation of biotin tripeptide-1:
adding N, N '-carbonyl imidazole into Biotin-Gly-OH and tetrahydrofuran under an ice bath cooling condition, dropwise adding the N, N' -carbonyl imidazole into a tetrahydrofuran solution containing His (Trt) -Lys (Boc) -OH and triethylamine with the concentration of 9.2 wt% when a reaction solution does not bubble any more, reacting at room temperature after finishing dropping, monitoring the reaction by TLC, concentrating under reduced pressure after finishing the reaction, adding water and ethyl acetate, adjusting the pH to 2 by using hydrochloric acid with the concentration of 2M, washing an organic phase by using brine, drying anhydrous sodium sulfate, and concentrating under reduced pressure to obtain a product M; and then deprotection, namely adding dichloromethane into the product M under the protection of nitrogen to dissolve the product M, adding isovolumetric TFA into the product M, stirring the mixture at room temperature for 1 hour, monitoring the reaction by TLC, evaporating the solvent to dryness under reduced pressure at 55 ℃, washing the solvent for 4 times by diethyl ether, and drying the solvent in vacuum at 50 ℃ for 24 hours to obtain a product N, namely the biotin tripeptide-1; wherein the solid-to-liquid ratio of Biotin-Gly-OH to tetrahydrofuran is 1 g:10.5 mL; the mass ratio of Biotin-Gly-OH to N, N' -carbonyl imidazole is 1: 0.54; the mass ratio of His (Trt) -Lys (Boc) -OH to Biotin-Gly-OH is 1: 1.8; the mass ratio of triethylamine to Biotin-Gly-OH is 1: 0.41.1H NMR (400 MHz, CDCl3), δppm: 8.84 (s, 1H, IMI-H), 7.72 (s, 1H, IMI-H), 5.08、4.62~4.70、4.61 (4H, -CH), 3.44 (1H, S-CH), 3.25、3.02 (2H, IMI-CH2), 3.19、2.90 (2H, S-CH2), 4.15、2.71、2.23~1.40 (18H, -CH2)。HRMS (ESI): Calcd for C24H38N8O6S, m/z [M+H]+, 566.32。
Example 2:
the functionalized ionic liquid was prepared as in example 1.
A liquid phase synthesis method of biotin tripeptide-1 is different from the method of example 1 in that:
in the preparation process of His (Trt) -Lys (Boc) -OH, the molar ratio of the functionalized ionic liquid to the Boc-Lys-OH is 1: 1.9; the molar ratio of the ionophore A, Trt-His-OH was 1: 1.42; the solid-to-liquid ratio of the ionophore B to the sodium hydroxide was 1 g:0.84 mL.
In the preparation process of Biotin-Gly-OH, the molar ratio of Biotin-N-hydroxysuccinimide ester to glycine is 1: 1.12.
In the preparation process of the Biotin tripeptide-1, the mass ratio of His (Trt) -Lys (Boc) -OH to Biotin-Gly-OH is 1: 1.6.
Example 3:
the functionalized ionic liquid was prepared as in example 1.
A liquid phase synthesis method of biotin tripeptide-1 is different from the method of example 1 in that:
in the preparation process of His (Trt) -Lys (Boc) -OH, the molar ratio of the functionalized ionic liquid to the Boc-Lys-OH is 1: 2.35; the molar ratio of the ionophore A, Trt-His-OH was 1: 1.55; the solid-to-liquid ratio of the ionophore B to the sodium hydroxide was 1 g:0.94 mL.
In the preparation process of Biotin-Gly-OH, the molar ratio of Biotin-N-hydroxysuccinimide ester to glycine is 1: 1.26.
In the preparation process of the Biotin tripeptide-1, the mass ratio of His (Trt) -Lys (Boc) -OH to Biotin-Gly-OH is 1: 1.85.
Example 4:
the functionalized ionic liquid was prepared as in example 1.
A liquid phase synthesis method of biotin tripeptide-1 is different from the method of example 1 in that:
in the preparation process of His (Trt) -Lys (Boc) -OH, the molar ratio of the functionalized ionic liquid to the Boc-Lys-OH is 1: 2.4; the molar ratio of the ionophore A, Trt-His-OH is 1: 1.6; the solid-to-liquid ratio of the ionophore B to the sodium hydroxide was 1 g:1 mL.
In the preparation process of Biotin-Gly-OH, the molar ratio of Biotin-N-hydroxysuccinimide ester to glycine is 1: 1.28.
In the preparation process of the Biotin tripeptide-1, the mass ratio of His (Trt) -Lys (Boc) -OH to Biotin-Gly-OH is 1: 1.96.
Example 5:
the functionalized ionic liquid was prepared as in example 1.
A liquid phase synthesis method of biotin tripeptide-1 is different from the method of example 1 in that: during the preparation of His (Trt) -Lys (Boc) -OH, further purification was performed using a functionalized macroporous resin.
The method specifically comprises the following steps: during the preparation process of His (Trt) -Lys (Boc) -OH, the obtained ionophore B is dissolved in 60% ethanol solution, and the concentration is 0.04 g/mL; adding functional macroporous resin (solid-to-liquid ratio of 1 g:55 mL) for adsorption for 3h, and then desorbing, namely putting the adsorbed resin into 30% ethanol solution, oscillating for 30 min, filtering, distilling under reduced pressure, vacuum drying, and then performing ionic liquid carrier cutting.
Wherein, the preparation of the functional macroporous resin comprises the following steps:
taking macroporous adsorption resin of a poly (GMA-co-EDMA) matrix, 4-amino-4-deoxy-10-methylpteroic acid, M-phenylenediamine and M-phthalaldehyde according to the mass ratio of 1:0.22:0.16:0.18, mixing and dispersing in dioxane, adding an acetic acid aqueous solution with the concentration of 3M, placing the system in an oil bath condition at 90 ℃, and stirring and reacting for 24 hours in a nitrogen atmosphere; centrifuging, collecting precipitate, washing with THF and methanol for 5 times, and vacuum drying at 120 deg.C overnight to obtain functional macroporous resin; wherein the solid-to-liquid ratio of the macroporous adsorption resin of the poly (GMA-co-EDMA) matrix to the dioxane is 16 mg/mL; the volume of the acetic acid aqueous solution added was 3.2% of that of dioxane.
Example 6:
the functionalized ionic liquid was prepared as in example 1.
A liquid phase synthesis method of biotin tripeptide-1 is different from the method of example 1 in that: during the preparation process of the biotin tripeptide-1, the biotin tripeptide-1 is further purified by adopting functionalized macroporous resin.
The method specifically comprises the following steps: in the preparation process of the biotin tripeptide-1, the obtained product N is dissolved in a 60% ethanol solution, and the concentration is 0.04 g/mL; adding functional macroporous resin (solid-to-liquid ratio of 1 g:55 mL) for adsorption for 3h, and then desorbing, i.e. putting the adsorbed resin into 30% ethanol solution, oscillating for 30 min, filtering, distilling under reduced pressure, and vacuum drying to obtain biotin tripeptide-1.
Wherein, the preparation of the functionalized macroporous resin is the same as that of the embodiment 5.
Example 7:
the functionalized ionic liquid was prepared as in example 1.
A liquid phase synthesis method of biotin tripeptide-1 is different from the method of example 1 in that: during the preparation of His (Trt) -Lys (Boc) -OH and during the preparation of biotin tripeptide-1, further purification was performed using functionalized macroporous resins. The purification procedure was the same as in example 5 and example 6, respectively; the functionalized macroporous resin was prepared as in example 5.
Example 8:
the functionalized ionic liquid was prepared differently from example 5 in that: 4-chloro-1-butanol is used to replace 2-chloro-6, 7-dimethoxy-3H-quinazolin-4-one.
A liquid phase synthesis method of biotin tripeptide-1 is different from the method of example 5 in that: the functionalized ionic liquid prepared in this example was used.
Example 9:
the functionalized ionic liquid was prepared differently from example 6 in that: 4-chloro-1-butanol is used to replace 2-chloro-6, 7-dimethoxy-3H-quinazolin-4-one.
A liquid phase synthesis method of biotin tripeptide-1 is different from the method of example 6 in that: the functionalized ionic liquid prepared in this example was used.
Example 10:
the functionalized ionic liquid was prepared differently from example 7 in that: 4-chloro-1-butanol is used to replace 2-chloro-6, 7-dimethoxy-3H-quinazolin-4-one.
A liquid phase synthesis method of biotin tripeptide-1 is different from the method of example 7 in that: the functionalized ionic liquid prepared in this example was used.
Example 11:
the functionalized ionic liquid was prepared as in example 10.
A liquid phase synthesis method of biotin tripeptide-1 is different from the method of example 10 in that: in the preparation process of the functionalized macroporous resin, m-phenylenediamine is adopted to replace 4-amino-4-deoxy-10-methylpteroic acid.
Comparative example 1:
the functionalized ionic liquid was prepared differently from example 1 in that: 4-chloro-1-butanol is used to replace 2-chloro-6, 7-dimethoxy-3H-quinazolin-4-one.
The difference between the liquid phase synthesis method of the biotin tripeptide-1 and the example 1 is that the functionalized ionic liquid prepared by the comparative example is adopted.
Test example 1:
1. infrared characterization
The test adopts VERTEX 70 type Fourier transform infrared spectrum produced by BRUKER company in Germany, and the test is carried out by a potassium bromide tabletting method, and the ionic liquid sample is uniformly smeared on the tabletting by utilizing a sample application capillary. The test wavelength is 4000-500 cm-1Resolution of 4 cm-1And 16 times.
The functionalized ionic liquids prepared in comparative example 1 and example 1 were subjected to the above-mentioned tests, and the results are shown in fig. 1. From the analysis in the figure, compared with the infrared test result of the functionalized ionic liquid prepared in the comparative example 1, the infrared spectrum of the functionalized ionic liquid prepared in the example 1 is 1600-1500 cm-1The characteristic absorption peak of benzene ring skeleton vibration appears in the range, which indicates that the functionalized ionic liquid in the example 1 is successfully prepared.
The functionalized macroporous resins prepared in example 5 and example 11 were subjected to the above tests, and the results are shown in fig. 2. From the figureAnalysis revealed that the IR spectrum of the functionalized macroporous resin prepared in example 5 was 1675 cm, compared to the IR test result of the functionalized macroporous resin prepared in example 11-1A characteristic absorption peak of C = O bond in carboxyl group appeared nearby, in 1625 cm-1The adjacent peak signals are obviously enhanced and are caused by the characteristic absorption peak of an acetal imine bond, which shows that 4-amino-4-deoxy-10-methyl pteroic acid and m-phthalaldehyde have gradual polymerization reaction, and other group peak signals are covered in the macroporous absorption resin peak signals of a poly (GMA-co-EDMA) matrix; the above results indicate the successful preparation of the functionalized macroporous resin in example 5.
2. Determination of purity and yield
The test was performed by high performance liquid chromatography. Dissolving a biotin tripeptide-1 sample in water, wherein the concentration is 0.5 mg/mL; the test conditions specifically include: c18Chromatography column (4.6X 250 mm); the mobile phase, the phase A is trifluoroacetic acid/water with the volume ratio of 0.2 percent, and the phase B is trifluoroacetic acid/acetonitrile with the volume ratio of 0.1 percent; flow rate, 1.0 mL/min; sample size, 10 μ L; elution gradient, wherein the concentration of the phase B is 20-70%; elution time, 30 min.
The results of the above tests on the biotin tripeptide-1 synthesized in comparative example 1 and examples 1 to 11 are shown in Table 1:
TABLE 1 purity and yield test results
As can be seen from the data in Table 1, the purity and yield of the biotin tripeptide synthesized in example 1 are obviously higher than those of comparative example 1, which shows that the functionalized ionic liquid prepared by modifying 2-chloro-6, 7-dimethoxy-3H-quinazolin-4-one is used as a carrier for polypeptide liquid synthesis, so that the purity and yield of the biotin tripeptide-1 product can be effectively improved, the separation and purification operations of each stage are simple, further chromatographic purification is not needed, and the production cost is reduced. Example 5 the purity and yield of the synthesized biotin tripeptide-1 are obviously higher than example 1, and the purity of the synthesized biotin tripeptide-1 in example 6 is higher than example 1, and the yield is equivalent to that of example 1, which shows that the intermediate product is further separated and purified by adding the 4-amino-4-deoxy-10-methylpterinic acid modified functionalized macroporous resin in the liquid phase synthesis process of the biotin tripeptide-1, so that the purity and yield of the synthesized biotin tripeptide-1 product can be further increased; the separation and purification of the functionalized macroporous resin are further carried out after the finally obtained product, so that the purity of the synthesized product can be further improved, and the yield is not obviously influenced; the same trends for examples 8 and 9 compared to comparative example 1 are consistent with the above. In addition, the effect of example 10 is better than that of example 11, which shows that the separation and purification effect of the 4-amino-4-deoxy-10-methylpteroic acid modified functionalized macroporous resin is better.
Test example 2:
functionalized macroporous resin characterization
1. Measurement of adsorption-desorption Performance
Static adsorption test
Pretreating, namely soaking the functionalized macroporous resin in absolute ethyl alcohol for 24 hours, fully swelling, then carrying out column filling, allowing absolute ethyl alcohol with the volume 5 times of that of a bed layer to pass through the resin at the flow rate of 3 BV/h until an absorption peak does not exist at the position where a sample peak appears, and then washing with deionized water at the flow rate of 4 BV/h until no ethanol smell exists for later use;
50 g of the resin sample which is pre-treated is taken, 100 mL of biotin tripeptide-1 alcohol solution with the concentration of 0.2 g/mL (the ethanol concentration is 60%) is added, the mixture is sealed and then put into a constant temperature shaking table at 20 ℃ for 3 hours with the frequency of 140 times/min, and then the mixture is taken out and kept stand for 3 hours. Stirring once every half hour to ensure complete adsorption. The solvent was then filtered and washed with 50 mL of deionized water, the filtrate was concentrated, freeze dried, weighed, and the adsorption equivalent was calculated according to the following formula:
adsorption equivalent weight (mg/g) = adsorption amount/dry resin mass
Static desorption test
Taking a dry solid resin sample obtained after static adsorption, adding ethanol with the concentration of 40%, sealing, and then placing on a constant temperature shaking table at 20 ℃ for oscillation for 3h at the frequency of 140 times/min. Then taking out, carrying out suction filtration on the solvent, concentrating the filtrate, freeze-drying, weighing, and calculating the desorption rate according to the following formula:
desorption% = desorption/adsorption × 100%
The above tests were performed on the functionalized macroporous resins prepared in example 5 and example 11, and the results are shown in table 2:
TABLE 2 adsorption-desorption Performance test results
As can be seen from the data in table 2, the adsorption equivalent and desorption rate of the functionalized macroporous resin prepared in example 5 are significantly higher than those of example 11, which indicates that the adsorption performance of the macroporous resin can be significantly enhanced and the adsorption equivalent is significantly increased by using the modified functionalized macroporous resin modified by 4-amino-4-deoxy-10-methylpteroic acid; and the desorption performance is also obviously enhanced, and the desorption rate is obviously increased.
2. Stability determination
The experiment was carried out by taking 50 g of a sample of the resin which had been previously treated, and conducting the adsorption-desorption operation 10 times, the specific operation method being the same as the above-mentioned measurement of the adsorption-desorption performance, and the result being characterized by the reduction rate of the adsorption equivalent.
The above tests were performed on the functionalized macroporous resins prepared in example 5 and example 11, and the results are shown in table 3:
table 3 stability test results
As can be seen from the data in Table 3, the reduction rate of the adsorption equivalent of the functionalized macroporous resin prepared in example 5 is obviously lower than that of example 11, which shows that the stability of the macroporous resin can be effectively improved, the macroporous resin can be reused for many times, and the service life of the macroporous resin can be effectively prolonged by adopting the functionalized macroporous resin modified by 4-amino-4-deoxy-10-methylpteroic acid.
3. Selective adsorption assay
An alcohol solution of Biotin tripeptide-1 (prepared in example 1), an alcohol solution of ionophore B (prepared in example 1), an alcohol solution of functionalized ionic liquid (prepared in example 1), an alcohol solution of His (Trt) -Lys (Boc) -OH (prepared in example 1), and an alcohol solution of Biotin-Gly-OH (prepared in example 1) were prepared at a concentration of 0.02 g/mL, respectively, and 5 g of the functionalized macroporous resin was added to perform the adsorption-desorption experiments, which were performed as described above.
The above tests were performed on the functionalized macroporous resins prepared in example 5 and example 11, and the results are shown in table 4:
TABLE 4 adsorption-desorption Performance test results
As can be seen from the data in Table 4, the adsorption equivalent of the functionalized macroporous resin prepared in example 5 on the biotin tripeptide-1 and the ionophore B is obviously higher than that on other substances, and the functionalized macroporous resin has good selective adsorption; and the adsorption amount of the functionalized ionic liquid, His (Trt) -Lys (Boc) -OH and Biotin-Gly-OH is obviously less than that of the ionic liquid prepared in example 11; the method shows that the functional macroporous resin modified by the 4-amino-4-deoxy-10-methylpteroic acid can effectively improve the selective adsorption capacity of the macroporous resin on the biotin tripeptide-1 and the ionophore B, and achieve better separation and purification effects.
Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, will not be described in detail herein.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (7)
1. A liquid phase synthesis method of biotin tripeptide-1 comprises the following steps:
preparation of His (Trt) -Lys (Boc) -OH:
mixing, stirring and dissolving a functionalized ionic liquid, Boc-Lys-OH, DMAP and acetonitrile, adding a DCC/dichloromethane solution with the concentration of 0.8-1M, stirring and reacting for 24-28 h under the conditions of room temperature and nitrogen protection, performing suction filtration by using a Buchner funnel paved with two layers of filter paper and diatomite, performing reduced pressure distillation and ether washing, dissolving in dichloromethane, washing with hydrochloric acid with the concentration of 1.8-2M, adding anhydrous sodium sulfate into an organic phase, drying, performing reduced pressure distillation and concentration, and performing vacuum drying at 40-50 ℃ for 24-30 h to obtain an ionophore A;
mixing an ionophore A, PyBOP, Trt-His-OH, DIPEA and acetonitrile, stirring and dissolving under the protection of nitrogen, stirring and reacting for 8-10 h under the condition of constant-temperature water bath at 35-40 ℃, distilling under reduced pressure at 50-55 ℃ to remove a solvent, sequentially washing with diethyl ether and deionized water for 3-4 times respectively, adding anhydrous sodium sulfate, drying, and vacuum drying for 24-30 h to obtain an ionophore B;
taking 0.8-1M sodium hydroxide, THF/water (v/v, 1: 1.5-2.5) and an ionophore B, stirring at room temperature under the protection of nitrogen, carrying out TLC monitoring reaction, carrying out reduced pressure distillation at 50-55 ℃, adding 1.8-2M hydrochloric acid to acidify until the pH is 5-6, filtering, washing precipitates for 2-3 times with deionized water, and carrying out vacuum drying at 70-80 ℃ for 24-30 h to obtain His (Trt) -Lys (Boc) -OH;
preparation of Biotin-Gly-OH:
-Biotin-Gly-OH is obtained by chemical reaction of Biotin-N-hydroxysuccinimide ester with glycine;
preparation of biotin tripeptide-1:
his (Trt) -Lys (Boc) -OH and Biotin-Gly-OH are subjected to dehydration condensation reaction and deprotection to obtain Biotin tripeptide-1;
wherein the functionalized ionic liquid structure at least comprises a hydroxyl active group; the raw materials of the functionalized ionic liquid at least comprise 2-chloro-6, 7-dimethoxy-3H-quinazoline-4-ketone and 1-methylimidazole;
the preparation method of the functionalized ionic liquid comprises the following steps:
mixing 1-methylimidazole and 2-chloro-6, 7-dimethoxy-3H-quinazoline-4-ketone, heating to 80-90 ℃, and reacting in a nitrogen atmosphere to obtain the compound.
2. The liquid phase synthesis method of biotin tripeptide-1 according to claim 1, characterized in that: the purity of the biotin tripeptide-1 is more than or equal to 96 percent, and the yield is more than 90 percent.
3. The liquid phase synthesis method of biotin tripeptide-1 according to claim 1, characterized in that: during the preparation process of His (Trt) -Lys (Boc) -OH and/or during the preparation process of biotin tripeptide-1, further purifying by adopting functionalized macroporous resin; wherein, the raw materials of the functional macroporous resin comprise macroporous absorption resin of poly (GMA-co-EDMA) matrix, 4-amino-4-deoxy-10-methylpteroic acid, m-phenylenediamine and m-phthalaldehyde.
4. The liquid phase synthesis method of biotin tripeptide-1 according to claim 1, characterized in that: the molar ratio of the 1-methylimidazole to the 2-chloro-6, 7-dimethoxy-3H-quinazoline-4-one is 1: 1.2-1.6.
5. Use of the functionalized ionic liquid of claim 1 as a support material, catalyst.
6. The use of the functionalized ionic liquid of claim 1 in the field of liquid phase synthesis of polypeptides.
7. The use according to claim 6, wherein the functionalized ionic liquid is used for improving the purity and yield of polypeptide products.
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