Method for preparing Fmoc-Ile-OH through chemical derivation

Abstract

The invention discloses a method for preparing Fmoc-Ile-OH through chemical derivation, and relates to the technical field of polypeptide solid-phase synthesis. The specific preparation method comprises the following steps: purifying an Ile derivative, adding a protecting group reagent, an alkaline agent and a solvent, reacting, and purifying to obtain an intermediate; mixing the intermediate, a catalyst and a solvent, introducing a hydrogenation reagent, reacting, and purifying to obtain Fmoc-Ile-OH; or mixing the intermediate and a demethylation reaction reagent, reacting, and purifying to obtain Fmoc-Ile-OH. The preparation method provided by the invention can potentially avoid the problem in polypeptide synthesis caused by trace Fmoc-Val-OH and Fmoc-Nva-OH in Fmoc-Ile-OH, and the conversion rate of the intermediate and the yield of Fmoc-Ile-OH in the preparation method provided by the invention are relatively high, so that the preparation method provided by the invention has a wide application prospect in synthesis of Ile-containing polypeptide drugs.

Description

Technical Field

[0001] The invention relates to the technical field of polypeptide solid phase synthesis, and in particular to a method for preparing Fmoc-Ile-OH by chemical derivatization. Background Art

[0002] The solid phase synthesis of peptides usually adopts the strategy of step-by-step coupling of amino acids, and the amino acids protected by Fmoc amino groups used have been commercialized. Among them, isoleucine is prepared by biological fermentation. In the process of fermentation to prepare isoleucine, specific microbial strains are usually used for fermentation. These strains will produce a variety of intermediates during metabolism, among which the synthesis pathways of valine (Val), norvaline (Nva) and isoleucine (Ile) are intersecting. Specifically, the synthesis pathways of isoleucine and valine overlap in multiple steps, resulting in the inevitable production of valine and norvaline in the process of synthesizing isoleucine. The above two types of amino acids are similar in structure to isoleucine and have similar properties. In the synthesis of peptide drugs, the impurities they produce are difficult to separate from the target product in reverse phase chromatography, resulting in difficulties in the purification of peptides, high purification difficulty and high purification cost, which affects the yield of peptide synthesis. Therefore, it is necessary to develop an efficient and convenient method for purifying isoleucine to eliminate impurities that are difficult to remove due to a small amount of valine (Val) and norvaline (Nva) in the starting material, reduce the number of peptide purifications, reduce the difficulty of purification, and improve the purification yield of peptides. Summary of the invention

[0003] The object of the present invention is to provide a method for preparing Fmoc-Ile-OH by chemical derivatization, so as to solve the problems of difficult preparation and purification and many impurities in the existing preparation of Fmoc-Ile-OH.

[0004] In order to solve the above technical problems, the present invention specifically provides the following technical solutions: The present invention discloses a method for preparing Fmoc-Ile-OH by chemical derivatization, comprising: S1: purifying the Ile derivative first, then adding a protecting group reagent, a base agent and a solvent to react and purify to obtain an intermediate; S2: mixing the intermediate, the catalyst and the solvent, introducing a hydrogenation reagent to react and purifying to obtain Fmoc-Ile-OH; or mixing the intermediate and a demethylation reaction reagent to react and purifying to obtain Fmoc-Ile-OH; the mass ratio of the intermediate to the catalyst used is 1:0.15-0.8.

[0005] Preferably, the catalyst in S2 is at least one of palladium carbon and loaded metal carbon, and the loaded metal carbon is prepared from grapefruit peel, 2,2-bipyridyl N-oxide, 1-chloro-4-(trifluoromethyl)benzene, palladium acetate and sodium borohydride. The loaded metal carbon has a good catalytic effect, can effectively promote the activation of the hydrogenation reagent, and make the intermediate and the hydrogenation reagent react efficiently. At the same time, grapefruit peel, 2,2-bipyridyl N-oxide, 1-chloro-4-(trifluoromethyl)benzene are used to prepare doped activated carbon, and the loaded metal carbon is further loaded with metal, which can improve the selectivity of the loaded metal carbon, thereby improving the conversion rate of the intermediate, the yield of Fmoc-Ile-OH and reducing the impurity content.

[0006] Preferably, the Ile derivative in S1 is at least one of H-Ile-Obzl and H-Ile-OMe; H-Ile-OMe is prepared from L-isoleucine methyl ester hydrochloride, 2-hydroxy-4-methoxybenzaldehyde and ammonium formate.

[0007] Preferably, the protecting group reagent in S1 is Fmoc-Osu, and the mass ratio of the used amounts of the Ile derivative and the protecting group reagent is 1:1-3.

[0008] Preferably, the alkaline agent in S1 is sodium carbonate, and the mass ratio of the Ile derivative to the alkaline agent is 1:0.4-1.8.

[0009] Preferably, the solvent in S1 is a tetrahydrofuran aqueous solution, which consists of tetrahydrofuran and water, the volume ratio of tetrahydrofuran to water is 1:0.8-1.2, and the ratio of Ile derivative to solvent is 1:10-30 ml.

[0010] Preferably, the solvent in S2 is methanol, and the usage ratio of the intermediate to methanol is 1 g: 4-10 ml.

[0011] Preferably, the hydrogenation agent in S2 is hydrogen.

[0012] Preferably, the demethylation reaction reagents in S2 are calcium chloride solution and sodium hydroxide solution, the calcium chloride solution consists of calcium chloride, isopropanol and water, the dosage ratio of calcium chloride to isopropanol is 1g:10-20ml, the volume ratio of isopropanol to water is 1:0.2-0.8, the dosage ratio of the intermediate to the calcium chloride solution is 1g:50-60ml, the sodium hydroxide solution consists of sodium hydroxide and water, the dosage ratio of sodium hydroxide to water is 1g:40-60ml, and the dosage ratio of Fmoc-Ile-OMe to the sodium hydroxide solution is 1g:8-15ml.

[0013] Preferably, the reaction temperature in S1 is 20-30°C, and the reaction is detected by TLC.

[0014] Preferably, the reaction temperature of introducing the hydrogenation reagent into S2 is 20-30°C, the reaction time is 3-5h, and the mixed reaction temperature of the intermediate and the demethylation reaction reagent is 20-30°C, and the reaction time is 3-5h.

[0015] The invention discloses Fmoc-Ile-OH prepared by the preparation method.

[0016] The present invention discloses a preparation method for purifying H-(Hmb)Ile-OMe, which is specifically: L-isoleucine methyl ester hydrochloride and dichloromethane are mixed, and then a saturated sodium bicarbonate solution is added, and the organic phase is separated and dried to obtain a concentrated solution. 2-Hydroxy-4-methoxybenzaldehyde and sodium triacetoxyborohydride are added to the concentrated solution, and the reaction is detected by TLC at 20-30° C. After the reaction is completed, quenching, extraction and concentration are performed to obtain a residual sample, and the residual sample is separated by column chromatography to obtain H-(Hmb)Ile-OMe, and the H-(Hmb)Ile-OMe is dissolved and eluted and freeze-dried to obtain purified H-(Hmb)Ile-OMe.

[0017] Preferably, the usage ratio of L-isoleucine methyl ester hydrochloride to dichloromethane is 1 g: 20-30 ml.

[0018] Preferably, the mass ratio of L-isoleucine methyl ester hydrochloride to 2-hydroxy-4-methoxybenzaldehyde is 1:0.8-1.2.

[0019] Preferably, the mass ratio of L-isoleucine methyl ester hydrochloride to sodium triacetoxyborohydride is 1:1-2.

[0020] Preferably, the solvent used for quenching is a saturated sodium bicarbonate solution, which consists of sodium bicarbonate and water.

[0021] Preferably, the solvent used for extraction is dichloromethane.

[0022] Preferably, the solution used to dissolve H-(Hmb)Ile-OMe is an acetonitrile solution, which is composed of acetonitrile and water, and the volume ratio of acetonitrile to water is 1:8-10.

[0023] Preferably, the eluent used for elution consists of formic acid, water and acetonitrile, the mass ratio of formic acid to water is 1:300-350, and the mass ratio of acetonitrile to water is 1:3-5.

[0024] The present invention discloses a method for preparing H-Ile-OMe, which is specifically: Purified H-(Hmb)Ile-OMe, palladium carbon, ammonium formate and methanol aqueous solution were mixed and reacted at 20-30°C for 10-15h under nitrogen. After the reaction, the filtrate was evaporated to obtain a residual liquid, and the residual liquid was separated by column chromatography to obtain H-Ile-OMe.

[0025] Preferably, the mass ratio of the amount of purified H-(Hmb)Ile-OMe to palladium carbon used is 1:0.1-0.5.

[0026] Preferably, the mass ratio of the amount of purified H-(Hmb)Ile-OMe to the amount of ammonium formate used is 1:0.5-0.3.

[0027] Preferably, the methanol aqueous solution consists of methanol and water, and the volume ratio of methanol to water is 1:0.1-0.5.

[0028] Preferably, the ratio of purified H-(Hmb)Ile-OMe to methanol aqueous solution is 1 g: 15-25 ml.

[0029] The present invention discloses a method for preparing Fmoc-Ile-OH by chemical derivatization, comprising: S1: Mixing the Ile derivative and the solvent, then adding the protecting group reagent and the alkaline agent, detecting the reaction by TLC at 20-30°C, and after the reaction, rotary evaporating and separating the aqueous phase, extracting the aqueous phase to separate the organic phase, and concentrating and separating the organic phase by column chromatography to obtain the intermediate; S2: Mix the intermediate and the demethylation reaction reagent, react at 20-30°C for 3-5h, add a reagent to separate the aqueous phase after the reaction, acidify the aqueous phase to pH 5-6.5, add an extractant to separate the organic phase, and finally wash, concentrate and separate by column chromatography to obtain Fmoc-Ile-OH.

[0030] Preferably, the Ile derivative in S1 is H-Ile-OMe.

[0031] Preferably, the protecting group reagent in S1 is Fmoc-Osu, and the mass ratio of the used amounts of the Ile derivative and the protecting group reagent is 1:1-3.

[0032] Preferably, the alkaline agent in S1 is sodium carbonate, and the mass ratio of the Ile derivative to the alkaline agent is 1:0.4-1.8.

[0033] Preferably, the solvent in S1 is a tetrahydrofuran aqueous solution, which consists of tetrahydrofuran and water, the volume ratio of tetrahydrofuran to water is 1:0.8-1.2, and the ratio of Ile derivative to solvent is 1:10-30 ml.

[0034] Preferably, the reagent used for extraction in S1 is dichloromethane.

[0035] Preferably, the demethylation reaction reagents in S2 are calcium chloride solution and sodium hydroxide solution.

[0036] More preferably, the calcium chloride solution consists of calcium chloride, isopropanol and water.

[0037] More preferably, the usage ratio of calcium chloride to isopropanol is 1 g:10-20 ml, and the volume ratio of isopropanol to water is 1:0.2-0.8.

[0038] More preferably, the usage ratio of the intermediate to the calcium chloride solution is 1 g: 50-60 ml.

[0039] More preferably, the sodium hydroxide solution consists of sodium hydroxide and water, and the dosage ratio of sodium hydroxide to water is 1g:40-60ml.

[0040] More preferably, the ratio of Fmoc-Ile-OMe to sodium hydroxide solution is 1 g: 8-15 ml.

[0041] Preferably, the reagent in S2 is diethyl ether.

[0042] Preferably, the reagent used to acidify the aqueous phase in S2 is a hydrochloric acid solution, which consists of hydrochloric acid and water, and the dosage ratio of hydrochloric acid to water is 1g:25-30ml.

[0043] Preferably, the extractant in S2 is ethyl acetate.

[0044] The present invention discloses a method for preparing Fmoc-Ile-OH by chemical derivatization, comprising: S1: dissolving the Ile derivative, purifying it by column chromatography, eluting it and freeze-drying it. Then mixing the Ile derivative with a solvent, adding a protecting group reagent and an alkaline agent, detecting the reaction by TLC at 20-30°C, and after the reaction is completed, rotary evaporation and separating the aqueous phase, extracting the aqueous phase to separate the organic phase, and concentrating and separating the organic phase by column chromatography to obtain the intermediate; S2: The intermediate, the catalyst and the solvent are mixed, a hydrogenation reagent is introduced and the mixture is reacted at 20-30° C. for 3-5 hours, and then the filtrate is collected and separated by column chromatography to obtain Fmoc-Ile-OH.

[0045] Preferably, the Ile derivative in S1 is H-Ile-Obzl.

[0046] Preferably, the eluent used for elution in S1 consists of formic acid, water and acetonitrile, the mass ratio of formic acid to water is 1:300-350, and the mass ratio of acetonitrile to water is 1:3-5.

[0047] Preferably, the protecting group reagent in S1 is Fmoc-Osu, and the mass ratio of the used amounts of the Ile derivative and the protecting group reagent is 1:1-3.

[0048] Preferably, the alkaline agent in S1 is sodium carbonate, and the mass ratio of the Ile derivative to the alkaline agent is 1:0.4-1.8.

[0049] Preferably, the solvent in S1 is a tetrahydrofuran aqueous solution, which consists of tetrahydrofuran and water, the volume ratio of tetrahydrofuran to water is 1:0.8-1.2, and the ratio of Ile derivative to solvent is 1:10-30 ml.

[0050] Preferably, the reagent used for extraction in S1 is dichloromethane.

[0051] Preferably, the catalyst in S2 is at least one of palladium carbon and supported metal carbon.

[0052] Preferably, the mass ratio of the intermediate to the catalyst used in S2 is 1:0.15-0.8.

[0053] Preferably, the solvent in S2 is methanol, and the usage ratio of the intermediate to methanol is 1 g: 4-10 ml.

[0054] Preferably, the hydrogenation agent in S2 is hydrogen.

[0055] The present invention discloses a method for preparing doped activated carbon, which is specifically: The grapefruit peel is ground into grapefruit peel powder, potassium hydroxide, 2,2-dipyridyl N-oxide and 1-chloro-4-(trifluoromethyl)benzene are added and mixed evenly, and the mixture is carbonized at 750-850°C for 1.5-3h at a heating rate of 4-6°C / min under a nitrogen atmosphere to obtain a sample, which is then acid-washed and water-washed and finally dried to obtain doped activated carbon.

[0056] Preferably, the mass ratio of the grapefruit peel powder to the potassium hydroxide is 1:1-2.

[0057] Preferably, the mass ratio of the grapefruit peel powder to the 2,2-bipyridyl N-oxide is 1:0.3-0.7.

[0058] Preferably, the mass ratio of 2,2-bipyridyl N-oxide to 1-chloro-4-(trifluoromethyl)benzene is 1:0.3-2.

[0059] The present invention discloses a method for preparing metal-loaded carbon, which is specifically: Add the doped activated carbon to the palladium acetate solution, stir at 20-30°C for 3-5h, then separate the solid, add the mixed solution and sodium borohydride to the solid, stir at 20-30°C for another 3-5h, separate the solid, wash and dry to obtain the loaded metal carbon.

[0060] Preferably, the palladium acetate solution consists of palladium acetate and toluene.

[0061] Preferably, the usage ratio of palladium acetate to toluene is 1 g:350-450 ml.

[0062] Preferably, the dosage ratio of the doped activated carbon to the palladium acetate solution is 1 g: 5-10 ml.

[0063] Preferably, the mixed solution consists of toluene and ethanol, and the volume ratio of toluene to ethanol is 1:0.2-0.8.

[0064] Preferably, the dosage ratio of the doped activated carbon to the mixed solution is 1 g: 6-10 ml.

[0065] Preferably, the mass ratio of the doped activated carbon to the sodium borohydride is 1:0.02-0.06.

[0066] Preferably, the washing agent is ethanol.

[0067] More preferably, in the process of preparing the loaded metal carbon of the present invention, on the basis of using 2,2-dipyridyl N-oxide and 1-chloro-4-(trifluoromethyl)benzene to prepare the doped activated carbon, cyclodextrin derivatives can also be used. The cyclodextrin derivatives are used in conjunction with the doped activated carbon to prepare the loaded metal carbon, which can further improve the activity and selectivity of the loaded metal carbon, improve the reaction efficiency of the intermediate and the hydrogenation reagent, and finally improve the conversion rate of the intermediate and the yield of Fmoc-Ile-OH, and the impurity content is also effectively reduced.

[0068] Preferably, the mass ratio of the doped activated carbon to the cyclodextrin derivative is 1:0.02-0.5.

[0069] The present invention discloses a method for preparing a cyclodextrin derivative, which is specifically: β-cyclodextrin, ethyl (chlorosulfonyl)acetate and tetrabutylammonium bromide are added to water, reacted at 20-30°C for 10-15h, then sodium hydroxide solution is added dropwise and the reaction is continued for 40-50min, ammonium chloride is added to the reaction filtrate to adjust the pH to 7.5-8.5, and the crude product is separated after standing at 0-5°C for 10-15h, and the crude product is washed, recrystallized and dried to obtain a cyclodextrin derivative.

[0070] Preferably, the mass ratio of β-cyclodextrin to ethyl (chlorosulfonyl)acetate is 1:0.2-0.3.

[0071] Preferably, the mass ratio of β-cyclodextrin to tetrabutylammonium bromide is 1:0.005-0.01.

[0072] Preferably, the dosage ratio of β-cyclodextrin to water is 1 g: 20-30 ml.

[0073] Preferably, the sodium hydroxide solution consists of sodium hydroxide and water, and the dosage ratio of sodium hydroxide to water is 1g:8-15ml.

[0074] Preferably, the usage ratio of β-cyclodextrin to sodium hydroxide solution is 1 g: 2-5 ml.

[0075] Compared with the prior art, the present invention has the following beneficial effects: The present invention proposes a method for preparing Fmoc-Ile-OH by chemical derivatization, wherein an Ile derivative is first purified, and then a protecting group reagent, an alkali agent and a solvent are added to react and purify to obtain an intermediate; then the intermediate, a catalyst and a solvent are mixed, a hydrogenation reagent is introduced to react and purify to obtain Fmoc-Ile-OH; or the intermediate and a demethylation reaction reagent are mixed to react and purify to obtain Fmoc-Ile-OH. The preparation method of the present invention can potentially avoid the problems in polypeptide synthesis caused by the presence of trace amounts of Fmoc-Val-OH and Fmoc-Nva-OH in Fmoc-Ile-OH, and the conversion rate of the intermediate and the yield of Fmoc-Ile-OH in the preparation method of the present invention are high, and the preparation method of the present invention has a wide application prospect in the synthesis of polypeptide drugs containing Ile. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0077] Figure 1 This is a SEM image of the doped activated carbon prepared in Example 3; Figure 2 It is the HPLC analysis chart of H-(Hmb)-Ile-OMe; Figure 3 HPLC analysis chart of purified H-(Hmb)-Ile-OMe; Figure 4 It is the HPLC analysis chart of H-Ile-Obzl; Figure 5 HPLC analysis chart of purified H-Ile-Obzl; Figure 6 This is the HPLC analysis chart of Fmoc-Ile-OH. DETAILED DESCRIPTION

[0078] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0079] The following first describes the concepts involved in the present application in conjunction with the accompanying drawings. It should be noted that the following description of each concept is only to make the content of the present application easier to understand, and does not limit the scope of protection of the present application; at the same time, the embodiments and features in the embodiments of the present application can be combined with each other in the absence of conflict. The present application will be described in detail with reference to the accompanying drawings and in conjunction with the embodiments.

[0080] Embodiment 1: Preparation of purified H-(Hmb)Ile-OMe: L-isoleucine methyl ester hydrochloride and dichloromethane are mixed, and then a saturated sodium bicarbonate solution is added, and the organic phase is separated and dried to obtain a concentrated solution. 2-Hydroxy-4-methoxybenzaldehyde and sodium triacetoxyborohydride are added to the concentrated solution, and the reaction is detected by TLC at 25°C. After the reaction is completed, a saturated sodium bicarbonate solution and dichloromethane are added for quenching and extraction, and the organic phase is separated and concentrated to obtain a residual sample. The residual sample is separated by column chromatography to obtain H-(Hmb)Ile-OMe, and an acetonitrile solution is added to H-(Hmb)Ile-OMe to dissolve, followed by elution with an eluent, and finally lyophilized to obtain purified H-(Hmb)Ile-OMe. The usage ratio of L-isoleucine methyl hydrochloride to dichloromethane is 1g:25ml, the mass ratio of L-isoleucine methyl hydrochloride to 2-hydroxy-4-methoxybenzaldehyde is 1:0.92, the mass ratio of L-isoleucine methyl hydrochloride to sodium triacetoxyborohydride is 1:1.7, the saturated sodium bicarbonate solution is composed of sodium bicarbonate and water, the acetonitrile solution is composed of acetonitrile and water, and the volume ratio of acetonitrile and water is 1:9, the eluent is composed of formic acid, water and acetonitrile, the mass ratio of formic acid to water is 1:332, and the mass ratio of acetonitrile to water is 1:4.

[0081] Preparation of H-Ile-OMe: Purified H-(Hmb)Ile-OMe, palladium carbon, ammonium formate and methanol aqueous solution are mixed, reacted at 25°C for 12 hours under nitrogen conditions, and the filtrate is evaporated to obtain a residual liquid after the reaction is completed. The residual liquid is separated by column chromatography to obtain H-Ile-OMe. The mass ratio of purified H-(Hmb)Ile-OMe to palladium carbon is 1:0.2, the mass ratio of purified H-(Hmb)Ile-OMe to ammonium formate is 1:0.9, the methanol aqueous solution is composed of methanol and water, the volume ratio of methanol and water is 1:0.25, and the amount ratio of purified H-(Hmb)Ile-OMe to methanol aqueous solution is 1g:20ml.

[0082] Preparation of Fmoc-Ile-OMe: H-Ile-OMe and tetrahydrofuran aqueous solution are mixed to obtain a mixed solution, Fmoc-Osu and sodium carbonate are added to the mixed solution, and the reaction is detected by TLC at 25°C. After the reaction is completed, the aqueous phase is separated by rotary evaporation, and dichloromethane is added to the aqueous phase to extract and separate the organic phase, and the organic phase is concentrated to obtain a concentrated solution, and the concentrated solution is separated by column chromatography to obtain Fmoc-Ile-OMe. The tetrahydrofuran aqueous solution is composed of tetrahydrofuran and water, and the volume ratio of tetrahydrofuran to water is 1:1, the amount ratio of H-Ile-OMe to tetrahydrofuran aqueous solution is 1:22.53 ml, the mass ratio of H-Ile-OMe to Fmoc-Osu is 1:2.56, and the mass ratio of H-Ile-OMe to sodium carbonate is 1:1.46.

[0083] Preparation of Fmoc-Ile-OH: Add calcium chloride solution and sodium hydroxide solution to Fmoc-Ile-OMe, react at 25°C for 4 hours, add ether to separate the aqueous phase after the reaction is completed, add hydrochloric acid solution to the aqueous phase to acidify to pH 6, then add ethyl acetate to extract and separate the organic phase, wash with saturated sodium chloride, dry and concentrate to obtain a crude product, and separate the crude product by column chromatography to obtain Fmoc-Ile-OH. The calcium chloride solution consists of calcium chloride, isopropanol and water, the dosage ratio of calcium chloride to isopropanol is 1g:16.09ml, the volume ratio of isopropanol to water is 1:0.43, the dosage ratio of Fmoc-Ile-OMe to calcium chloride solution is 1g:54.78ml, the sodium hydroxide solution consists of sodium hydroxide and water, the dosage ratio of sodium hydroxide to water is 1g:50ml, the dosage ratio of Fmoc-Ile-OMe to sodium hydroxide solution is 1g:10.96ml, and the hydrochloric acid solution consists of hydrochloric acid and water, the dosage ratio of hydrochloric acid to water is 1g:27.4ml.

[0084] Embodiment 2: Preparation of Fmoc-Ile-Obzl: Add water to H-Ile-Obzl to dissolve it, purify it by column chromatography, elute it with an eluent and freeze-dry it to obtain purified H-Ile-Obzl. Mix the purified H-Ile-Obzl, Fmoc-Osu, sodium carbonate and tetrahydrofuran aqueous solution, detect the reaction by TLC at 25°C, and after the reaction is completed, evaporate and separate the aqueous phase, add dichloromethane to the aqueous phase to extract and separate the organic phase, concentrate the organic phase to obtain a concentrated solution, and separate the concentrated solution by column chromatography to obtain Fmoc-Ile-Obzl. The eluent consists of formic acid, water and acetonitrile, the mass ratio of formic acid to water is 1:332, the mass ratio of acetonitrile to water is 1:4, the mass ratio of purified H-Ile-Obzl and Fmoc-Osu is 1:1.68, the mass ratio of purified H-Ile-Obzl and sodium carbonate is 1:0.48, the tetrahydrofuran aqueous solution consists of tetrahydrofuran and water, the volume ratio of tetrahydrofuran and water is 1:1, and the amount ratio of purified H-Ile-Obzl and tetrahydrofuran aqueous solution is 1:14.29 ml.

[0085] Preparation of Fmoc-Ile-OH: Fmoc-Ile-Obzl and palladium carbon were mixed, and then methanol was added under nitrogen, followed by hydrogen gas at 25°C for 4 hours. After the reaction, the filtrate was collected and separated by column chromatography to obtain Fmoc-Ile-OH. The mass ratio of Fmoc-Ile-Obzl to palladium carbon was 1:0.3, and the ratio of Fmoc-Ile-Obzl to methanol was 1g:6.5ml.

[0086] Embodiment 3: The preparation of Fmoc-Ile-Obzl is the same as in Example 2.

[0087] Preparation of doped activated carbon: Grind dried grapefruit peel into grapefruit peel powder, add potassium hydroxide, 2,2-dipyridyl N-oxide and 1-chloro-4-(trifluoromethyl)benzene and mix evenly, carbonize at 800℃ for 2h at a heating rate of 5℃ / min under nitrogen atmosphere to obtain a sample, wash with acid, then wash with water to pH 7, and finally dry to obtain doped activated carbon. The mass ratio of grapefruit peel powder to potassium hydroxide is 1:1.5, the mass ratio of grapefruit peel powder to 2,2-dipyridyl N-oxide is 1:0.5, and the mass ratio of 2,2-dipyridyl N-oxide to 1-chloro-4-(trifluoromethyl)benzene is 1:1.

[0088] Preparation of metal-loaded carbon: Add doped activated carbon to palladium acetate solution, stir at 25°C for 4 hours, separate solid, add mixed solution and sodium borohydride to the solid, stir at 25°C for another 4 hours, separate solid, wash with ethanol and dry to obtain metal-loaded carbon. The palladium acetate solution is composed of palladium acetate and toluene, the dosage ratio of palladium acetate to toluene is 1g:380ml, the dosage ratio of doped activated carbon to palladium acetate solution is 1g:8ml, the mixed solution is composed of toluene and ethanol, the volume ratio of toluene to ethanol is 1:0.5, the dosage ratio of doped activated carbon to mixed solution is 1g:8ml, and the mass ratio of doped activated carbon to sodium borohydride is 1:0.03.

[0089] Preparation of Fmoc-Ile-OH: Fmoc-Ile-Obzl and loaded metal carbon were mixed, and then methanol was added under nitrogen, followed by hydrogen gas at 25°C for 4 hours. After the reaction, the filtrate was collected and separated by column chromatography to obtain Fmoc-Ile-OH. The mass ratio of Fmoc-Ile-Obzl to loaded metal carbon was 1:0.3, and the ratio of Fmoc-Ile-Obzl to methanol was 1g:6.5ml.

[0090] Embodiment 4: The preparation of Fmoc-Ile-Obzl is the same as in Example 2.

[0091] Preparation of doped activated carbon: The preparation of doped activated carbon in this embodiment is compared with that in Example 3, except that the mass ratio of 2,2-dipyridyl N-oxide to 1-chloro-4-(trifluoromethyl)benzene is 1:1.5, and other conditions and parameters are the same as in Example 3.

[0092] The preparation of loaded metal carbon is the same as in Example 3.

[0093] The preparation of Fmoc-Ile-OH is the same as in Example 3.

[0094] Embodiment 5: The preparation of Fmoc-Ile-Obzl is the same as in Example 2.

[0095] Preparation of doped activated carbon: The preparation of doped activated carbon in this embodiment is compared with that in Example 3, except that the mass ratio of 2,2-dipyridyl N-oxide to 1-chloro-4-(trifluoromethyl)benzene is 1:0.6, and other conditions and parameters are the same as in Example 3.

[0096] The preparation of loaded metal carbon is the same as in Example 3.

[0097] The preparation of Fmoc-Ile-OH is the same as in Example 3.

[0098] Embodiment 6: The preparation of Fmoc-Ile-Obzl is the same as in Example 2.

[0099] The preparation of doped activated carbon is the same as in Example 3.

[0100] Preparation of cyclodextrin derivatives: β-cyclodextrin, ethyl (chlorosulfonyl) acetate and tetrabutylammonium bromide are added to water, reacted at 25°C for 12 hours, then sodium hydroxide solution is added dropwise and the reaction is continued for 45 minutes. After the reaction is completed, the filtrate is collected, ammonium chloride is added to adjust the pH to 8, and the crude product is separated after standing at 4°C for 12 hours. The crude product is washed with water and recrystallized and dried to obtain a cyclodextrin derivative. The mass ratio of β-cyclodextrin to ethyl (chlorosulfonyl) acetate is 1:0.26, the mass ratio of β-cyclodextrin to tetrabutylammonium bromide is 1:0.009, the mass ratio of β-cyclodextrin to water is 1g:24ml, the sodium hydroxide solution is composed of sodium hydroxide and water, the mass ratio of sodium hydroxide to water is 1g:10ml, and the mass ratio of β-cyclodextrin to sodium hydroxide solution is 1g:4ml.

[0101] Preparation of metal-loaded carbon: Mix the cyclodextrin derivative with water, then add the doped activated carbon, stir at 25°C for 2h, and separate to obtain pretreated doped activated carbon. Add the pretreated doped activated carbon to the palladium acetate solution, stir at 25°C for 4h, and then separate the solid. Add the mixed solution and sodium borohydride to the solid, stir at 25°C for another 4h, separate the solid, wash with ethanol and dry to obtain metal-loaded carbon. The dosage ratio of cyclodextrin derivative to water is 1g:500ml, the mass ratio of doped activated carbon to cyclodextrin derivative is 1:0.1, the palladium acetate solution is composed of palladium acetate and toluene, the dosage ratio of palladium acetate and toluene is 1g:380ml, the dosage ratio of pretreated doped activated carbon to palladium acetate solution is 1g:8ml, the mixed solution is composed of toluene and ethanol, the volume ratio of toluene and ethanol is 1:0.5, the dosage ratio of pretreated doped activated carbon to the mixed solution is 1g:8ml, and the mass ratio of pretreated doped activated carbon to sodium borohydride is 1:0.03.

[0102] Preparation of Fmoc-Ile-OH: The preparation of Fmoc-Ile-OH in this example is compared with that in Example 3, except that the loaded metal carbon is the loaded metal carbon prepared in this example, and the other conditions and parameters are the same as in Example 3.

[0103] Embodiment 7: The preparation of Fmoc-Ile-Obzl is the same as in Example 2.

[0104] The preparation of doped activated carbon is the same as in Example 3.

[0105] The preparation of cyclodextrin derivatives is the same as in Example 6.

[0106] Preparation of loaded metal carbon: The preparation of loaded metal carbon in the embodiment is compared with that in Example 6, except that the mass ratio of the doped activated carbon and the cyclodextrin derivative used is 1:0.3, and the other conditions and parameters are the same as in Example 6.

[0107] Preparation of Fmoc-Ile-OH: The preparation of Fmoc-Ile-OH in this example is compared with that in Example 6, except that the loaded metal carbon is the loaded metal carbon prepared in this example, and the other conditions and parameters are the same as in Example 6.

[0108] Embodiment 8: The preparation of Fmoc-Ile-Obzl is the same as in Example 2.

[0109] The preparation of doped activated carbon is the same as in Example 3.

[0110] The preparation of cyclodextrin derivatives is the same as in Example 6.

[0111] Preparation of loaded metal carbon: The preparation of loaded metal carbon in the embodiment is compared with that in Example 6, except that the mass ratio of the doped activated carbon and the cyclodextrin derivative used is 1:0.03, and the other conditions and parameters are the same as in Example 6.

[0112] Preparation of Fmoc-Ile-OH: The preparation of Fmoc-Ile-OH in this example is compared with that in Example 6, except that the loaded metal carbon is the loaded metal carbon prepared in this example, and the other conditions and parameters are the same as in Example 6.

[0113] Comparative Example 1: The preparation of Fmoc-Ile-Obzl is the same as in Example 2.

[0114] Preparation of doped activated carbon: The preparation of doped activated carbon in this embodiment is compared with that in Example 3, except that the mass ratio of 2,2-dipyridyl N-oxide to 1-chloro-4-(trifluoromethyl)benzene is 1:3, and other conditions and parameters are the same as in Example 3.

[0115] The preparation of loaded metal carbon is the same as in Example 3.

[0116] The preparation of Fmoc-Ile-OH is the same as in Example 3.

[0117] Comparative Example 2: The preparation of Fmoc-Ile-Obzl is the same as in Example 2.

[0118] Preparation of doped activated carbon: The preparation of doped activated carbon in this embodiment is compared with that in Example 3, except that the mass ratio of 2,2-dipyridyl N-oxide to 1-chloro-4-(trifluoromethyl)benzene is 1:0.05, and other conditions and parameters are the same as in Example 3.

[0119] The preparation of loaded metal carbon is the same as in Example 3.

[0120] The preparation of Fmoc-Ile-OH is the same as in Example 3.

[0121] Experimental Example 1: The morphology of the doped activated carbon prepared in Example 3 was observed by SEM microstructure. Figure 1 shown.

[0122] Experimental Example 2: HPLC analysis was performed on the materials used and the prepared Fmoc-Ile-OH. The materials used included H-(Hmb)-Ile-OMe and purified H-(Hmb)-Ile-OMe in Example 1, and H-Ile-Obzl and purified H-Ile-Obzl in Example 2. Fmoc-Ile-OH was the Fmoc-Ile-OH prepared in Example 2. Figure 2 is the HPLC analysis chart of H-(Hmb)-Ile-OMe, Figure 3 This is the HPLC analysis chart of purified H-(Hmb)-Ile-OMe. After purification, H-(Hmb)-Ile-OMe has only a single absorption peak, without obvious impurity peaks or interference from other components. Figure 4 is the HPLC analysis chart of H-Ile-Obzl, Figure 5 This is the HPLC analysis chart of purified H-Ile-Obzl, and the impurity peaks were removed after purification. Figure 6 This is the HPLC analysis chart of Fmoc-Ile-OH. The substance is single and has high purity and is a by-product.

[0123] Experimental Example 3: The yield of Fmoc-Ile-OH was statistically calculated for the Fmoc-Ile-OH prepared in Example 2-8 and Comparative Example 1-2. Yield = amount of Fmoc-Ile-OH generated / theoretical amount of Fmoc-Ile-OH generated × 100%.

[0124] Table 1 Fmoc-Ile-OH yield determination results

[0125] The results are shown in Table 1. Compared with Example 3, Example 2 shows that the use of supported metal carbon can improve the yield of Fmoc-Ile-OH; Compared with Example 4, Example 3 shows that the increase in the amount of 1-chloro-4-(trifluoromethyl)benzene within a certain range can improve the catalytic effect of the supported metal carbon finally prepared, and the yield of Fmoc-Ile-OH is improved; Compared with Example 5, Example 3 shows that the reduction in the amount of 1-chloro-4-(trifluoromethyl)benzene within a certain range can reduce the catalytic effect of the supported metal carbon finally prepared, that is, the yield of Fmoc-Ile-OH is reduced; Compared with Example 6, Example 3 shows that on the basis of using 2,2-bipyridyl N-oxide and 1-chloro-4-(trifluoromethyl)benzene to prepare doped activated carbon, adding cyclodextrin derivatives to prepare supported metal carbon can improve the supported metal carbon. The catalytic effect of metal carbon is improved, thereby improving the yield of Fmoc-Ile-OH; Example 6 is compared with Example 7, indicating that the increase in the amount of cyclodextrin derivatives used within a certain range can improve the catalytic effect of the prepared loaded metal carbon, thereby improving the yield of Fmoc-Ile-OH; Example 6 is compared with Example 8, indicating that the reduction in the amount of cyclodextrin derivatives used within a certain range will reduce the catalytic effect of the prepared loaded metal carbon, thereby reducing the yield of Fmoc-Ile-OH; Example 3 is compared with Comparative Examples 1 and 2, indicating that the usage ratio of 2,2-dipyridyl N-oxide and 1-chloro-4-(trifluoromethyl)benzene needs to be in an appropriate range, and too high or too low will have no obvious effect on the improvement of the catalytic effect of the loaded metal carbon finally prepared, that is, the yield of Fmoc-Ile-OH will not be significantly improved.

[0126] Experimental Example 4: The conversion rate of Fmoc-Ile-Obzl prepared into Fmoc-Ile-OH was statistically calculated for the Fmoc-Ile-OH prepared in Examples 2-8 and Comparative Examples 1-2. Conversion rate = molar mass of reacted Fmoc-Ile-Obzl / initial molar mass of Fmoc-Ile-Obzl × 100%.

[0127] Table 2 Fmoc-Ile-Obzl conversion rate determination results

[0128] The results are shown in Table 2. Compared with Example 3, Example 2 shows that the use of supported metal carbon can improve the conversion rate of Fmoc-Ile-Obzl; Compared with Example 4, Example 3 shows that the increase in the amount of 1-chloro-4-(trifluoromethyl)benzene within a certain range can improve the catalytic effect of the supported metal carbon finally prepared, and the conversion rate of Fmoc-Ile-Obzl is improved; Compared with Example 5, Example 3 shows that the reduction in the amount of 1-chloro-4-(trifluoromethyl)benzene within a certain range can reduce the catalytic effect of the supported metal carbon finally prepared, that is, the conversion rate of Fmoc-Ile-Obzl is reduced; Compared with Example 6, Example 3 shows that on the basis of using 2,2-bipyridyl N-oxide and 1-chloro-4-(trifluoromethyl)benzene to prepare doped activated carbon, adding cyclodextrin derivatives to prepare supported metal carbon can improve the supported metal carbon. The catalytic effect of the metal carbon is improved, thereby improving the conversion rate of Fmoc-Ile-Obzl; compared with Example 7, Example 6 shows that the increase in the amount of cyclodextrin derivatives used within a certain range can improve the catalytic effect of the prepared supported metal carbon, thereby improving the conversion rate of Fmoc-Ile-Obzl; compared with Example 8, Example 6 shows that the reduction in the amount of cyclodextrin derivatives used within a certain range will reduce the catalytic effect of the prepared supported metal carbon, thereby reducing the conversion rate of Fmoc-Ile-Obzl; compared with Comparative Examples 1 and 2, Example 3 shows that the usage ratio of 2,2-dipyridyl N-oxide and 1-chloro-4-(trifluoromethyl)benzene needs to be in a suitable range. Too high or too low a ratio has no obvious effect on the improvement of the catalytic effect of the supported metal carbon finally prepared, that is, the conversion rate of Fmoc-Ile-OH will not be significantly improved.

[0129] Experimental Example 5: Impurity content analysis: HPLC analysis was performed on the Fmoc-Ile-OH prepared in Example 2-8 and Comparative Example 1-2 to determine the impurity content.

[0130] Table 3 Impurity content determination results

[0131] The results are shown in Table 3. Compared with Example 3, Example 2 shows that the use of loaded metal carbon can reduce the impurity content; compared with Example 4, Example 3 shows that the increase in the amount of 1-chloro-4-(trifluoromethyl)benzene used within a certain range can result in better selectivity of the loaded metal carbon prepared and reduced impurity content; compared with Example 5, Example 3 shows that the reduction in the amount of 1-chloro-4-(trifluoromethyl)benzene used within a certain range can reduce the selectivity of the loaded metal carbon prepared, that is, increase the impurity content; compared with Example 6, Example 3 shows that on the basis of using 2,2-bipyridyl N-oxide and 1-chloro-4-(trifluoromethyl)benzene to prepare doped activated carbon, adding cyclodextrin derivatives to prepare loaded metal carbon can Improve the selectivity of loaded metal carbon, thereby reducing the impurity content; Example 6, compared with Example 7, shows that increasing the amount of cyclodextrin derivatives used within a certain range can improve the selectivity of the prepared loaded metal carbon, thereby reducing the impurity content; Example 6, compared with Example 8, shows that reducing the amount of cyclodextrin derivatives used within a certain range will reduce the selectivity of the prepared loaded metal carbon, thereby increasing the impurity content; Example 3, compared with Comparative Examples 1 and 2, shows that the usage ratio of 2,2-dipyridyl N-oxide and 1-chloro-4-(trifluoromethyl)benzene needs to be in an appropriate range, and too high or too low will have no obvious effect on the selectivity improvement of the loaded metal carbon finally prepared, that is, the impurity content will not be significantly reduced.

[0132] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any form. Any technical personnel in this field may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0133] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and its core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression and the objective existence of infinite specific structures, ordinary technicians in this technical field can make several improvements, modifications or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the concept and technical solution of the invention to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A method for preparing Fmoc-Ile-OH by chemical derivatization, comprising: S1: purifying the Ile derivative first, then adding a protecting group reagent, a base agent and a solvent to react and purify to obtain an intermediate; S2: mixing the intermediate, the catalyst and the solvent, introducing a hydrogenation reagent to react and purifying to obtain Fmoc-Ile-OH; or mixing the intermediate and a demethylation reaction reagent to react and purifying to obtain Fmoc-Ile-OH; the mass ratio of the intermediate to the catalyst used is 1:0.15-0.

8.

2. The method for preparing Fmoc-Ile-OH by chemical derivatization according to claim 1, characterized in that: The catalyst in S2 is at least one of palladium carbon and loaded metal carbon, and the loaded metal carbon is prepared from grapefruit peel, 2,2-dipyridyl N-oxide, 1-chloro-4-(trifluoromethyl)benzene, palladium acetate and sodium borohydride.

3. The method for preparing Fmoc-Ile-OH by chemical derivatization according to claim 1, characterized in that: The Ile derivative in S1 is at least one of H-Ile-Obzl and H-Ile-OMe; H-Ile-OMe is prepared from L-isoleucine methyl ester hydrochloride, 2-hydroxy-4-methoxybenzaldehyde and ammonium formate.

4. The method for preparing Fmoc-Ile-OH by chemical derivatization according to claim 1, characterized in that: The protecting group reagent in S1 is Fmoc-Osu, and the mass ratio of the used amount of Ile derivative and the protecting group reagent is 1:1-3.

5. The method for preparing Fmoc-Ile-OH by chemical derivatization according to claim 1, characterized in that: The alkali agent in S1 is sodium carbonate, and the mass ratio of the Ile derivative to the alkali agent is 1:0.4-1.

8.

6. The method for preparing Fmoc-Ile-OH by chemical derivatization according to claim 1, characterized in that: The solvent in S1 is tetrahydrofuran aqueous solution, which is composed of tetrahydrofuran and water. The volume ratio of tetrahydrofuran to water is 1:0.8-1.2, and the ratio of Ile derivative to solvent is 1:10-30 ml.

7. The method for preparing Fmoc-Ile-OH by chemical derivatization according to claim 1, characterized in that: The solvent in S2 is methanol, and the usage ratio of the intermediate to methanol is 1g:4-10ml.

8. The method for preparing Fmoc-Ile-OH by chemical derivatization according to claim 1, characterized in that: The hydrogenation reagent in S2 is hydrogen.

9. The method for preparing Fmoc-Ile-OH by chemical derivatization according to claim 1, characterized in that: The demethylation reaction reagents in S2 are calcium chloride solution and sodium hydroxide solution. The calcium chloride solution consists of calcium chloride, isopropanol and water. The dosage ratio of calcium chloride to isopropanol is 1g:10-20ml. The volume ratio of isopropanol to water is 1:0.2-0.

8. The dosage ratio of the intermediate to the calcium chloride solution is 1g:50-60ml. The sodium hydroxide solution consists of sodium hydroxide and water. The dosage ratio of sodium hydroxide to water is 1g:40-60ml. The dosage ratio of Fmoc-Ile-OMe to the sodium hydroxide solution is 1g:8-15ml.

10. Fmoc-Ile-OH prepared by the preparation method according to any one of claims 1 to 9.

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