Preparation method of Fmoc-Thr (tBu)-OH

Abstract

The invention discloses a preparation method of Fmoc-Thr (tBu)-OH. According to the method, L-threonine which is low in price and easy to obtain is used as a starting material, Tfa-Thr-OMe is prepared through methyl esterification and amino trifluoroacetylation protection, Tfa-Thr (tBu)-OMe and isobutene are subjected to a catalytic reaction to prepare Tfa-Thr (tBu)-OMe, then alkali saponification hydrolysis is performed, Fmoc protection is performed, and refining is performed to obtain a Fmoc-Thr (tBu)-OH finished product. The method has the advantages of simple process scheme, cheap and easily available raw materials, good reaction selectivity, easy operation and mild reaction conditions, and is suitable for large-scale production.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide drug synthesis, and in particular to a method for preparing Fmoc-Thr(tBu)-OH. Background Art

[0002] Fmoc-Thr(tBu)-OH, also known as fluorenylmethoxycarbonyl-L-threonine-tert-butyl ether, is an important intermediate in peptide synthesis. L-threonine is an essential amino acid widely used in pharmaceuticals, food, and animal feed. In recent years, demand for L-threonine has grown annually in both domestic and international markets. Fmoc-Thr(tBu)-OH, as a key protected amino acid, is used to construct sequences containing threonine residues, such as in the synthesis of peptide drugs like octreotide acetate, atosiban, semaglutide, and telpotide. In antibody-drug conjugates, threonine-containing peptide fragments are used for targeted drug delivery. Structurally, threonine has an additional methyl group compared to serine, which increases steric hindrance and, due to the electron-donating effect, renders the hydroxyl group of threonine less active. Using the same preparation strategy as serine, the conversion rate of threonine's hydroxyl group to ether is extremely low, making isolation and purification difficult and increasing costs. This makes the synthesis of doubly protected amino acids with Fmoc-Thr(tBu)-OH more challenging.

[0003] CN117551003 and Bulletin of the Chemical Society of Japan, 1982, vol. 55, # 9, pp. 3049–3050 disclose a method for preparing threonine tert-butyl ether by catalytic hydrogenation followed by the addition of an Fmoc protecting group. This method uses L-threonine as the starting material, undergoes methyl esterification, and protects the amino group with Cbz. The product is then reacted with isobutylene under concentrated sulfuric acid to produce benzyloxycarbonyl-threonine tert-butyl ether methyl ester (Cbz-Thr(tBu)-OMe). The Cbz protecting group is then removed under palladium-carbon hydrogenation or palladium-carbon ammonium formate conditions, followed by saponification to produce threonine tert-butyl ether. Finally, the product is protected with Fmoc using Fmoc-OSu to produce the final product, Fmoc-L-threonine tert-butyl ether. This process is long, multi-step, and complex. The cost of the precious metal catalyst is high, and the hydrogenation process is highly hazardous, making it unsuitable for large-scale industrial production.

[0004] CN109134314 discloses a non-hydrogenation route. This scheme uses L-threonine methyl ester hydrochloride as the starting material and catalyzes an etherification reaction in dichloromethane and isobutylene. Experimental verification found that under these process conditions, the reaction progressed slowly, almost completely. This may be due to the poor solubility of threonine hydrochloride under these conditions, resulting in a limited effective contact area for the reaction.

[0005] In summary, the existing process scheme has the disadvantages of harsh process conditions, high cost of raw and auxiliary materials, and low operability. Summary of the Invention

[0006] The present invention aims to provide a method for preparing Fmoc-Thr(tBu)-OH, thereby resolving the technical problems of harsh synthesis process conditions, high raw material costs, and unsuitability for large-scale production in the prior art. The method utilizes trifluoroacetyl-L-threonine methyl ester as a key intermediate protected amino acid, reacts with isobutylene under concentrated sulfuric acid catalysis, and the reaction proceeds to completion. After simple separation, the product is treated with alkali in situ to easily and simultaneously remove the trifluoroacetyl group and hydrolyze the methyl ester. The product is then reacted with the protective reagent Fmoc-OSu in a one-pot process to produce Fmoc-Thr(tBu)-OH. The crude product is recrystallized and purified to obtain a high-yield finished product. Compared with the prior art, this route boasts high conversion rates in each step, clean reactions, simple and mild reaction conditions, and the ability to achieve a one-pot continuous reaction, shortening the production cycle and improving efficiency, making it a process suitable for large-scale production.

[0007] To achieve the above objectives, the present invention provides the following technical solutions: The present invention provides a method for preparing Fmoc-Thr(tBu)-OH, comprising the following steps: S1, methyl esterification of L-threonine to prepare Tfa-Thr-OMe; S2, Tfa-Thr-OMe and isobutylene catalyze the reaction to prepare Tfa-Thr(tBu)-OMe; S3, alkaline saponification and hydrolysis of Tfa-Thr(tBu)-OMe to prepare H-Thr(tBu)-OH; Fmoc-Thr(tBu)-OH was prepared by Fmoc-protection on S4H-Thr(tBu)-OH; The route is as follows: .

[0008] Furthermore, the S1 reagent and reaction conditions are anhydrous methanol, dichlorothionine, and L-threonine, and the temperature is slowly raised to 30-35° C. and reacted for 24-48 hours.

[0009] Furthermore, the thionyl chloride can be replaced by thionyl chloride, hydrogen chloride methanol solution, trimethylsilyl chloride-methanol, etc.

[0010] Furthermore, S2 includes Tfa-Thr-OMe to prepare the intermediate trifluoroacetyl-L-threonine methyl ester, trifluoroacetyl-L-threonine methyl ester and isobutylene are catalyzed to prepare Tfa-Thr(tBu)-OMe, and the reagents and reaction conditions for preparing the intermediate trifluoroacetyl-L-threonine methyl ester from Tfa-Thr-OMe are anhydrous methanol, Tfa-Thr-Ome, triethylamine, and ethyl trifluoroacetate, and the temperature is slowly raised to 25°C and reacted for 12 to 24 hours.

[0011] Furthermore, the triethylamine can be replaced by other organic tertiary amines, such as trimethylamine and triisopropylamine.

[0012] Furthermore, the reagents and reaction conditions for the catalytic reaction of trifluoroacetyl-L-threonine methyl ester and isobutylene are trifluoroacetyl-L-threonine methyl ester, dichloromethane, concentrated sulfuric acid, isobutylene, and the reaction temperature is 0-5° C. for 12-20 hours.

[0013] Furthermore, the S3 is obtained by cooling the organic phase obtained in the previous step to 10-12° C., slowly adding sodium hydroxide solution to the system, and performing saponification reaction at 10-15° C. for 6 hours.

[0014] Furthermore, the sodium hydroxide can be replaced by other alkalis, such as potassium hydroxide.

[0015] Furthermore, the S4 is Fmoc-protected on H-Thr(tBu)-OH to obtain Fmoc-Thr(tBu)-OH, and the reagents and reaction conditions are sodium carbonate, acetone, Fmoc-Osu, and the reaction temperature is 25-35°C for 3 hours.

[0016] Based on the above technical solution, the embodiments of the present invention can produce at least the following technical effects: (1) The materials used in the present invention are all industrial products, the raw materials are cheap and easy to obtain, and the cost is low; (2) The process route used in the present invention involves conventional reactions, simple operation, mild reaction conditions and high safety; (3) The Tfa protecting group used in the present invention improves the selectivity of the reaction and the conversion rate of the substrate, simplifies the post-reaction treatment, and can achieve a one-pot continuous reaction; (4) The present invention avoids the use of expensive heavy metal catalysts and dangerous processes such as high-pressure hydrogenation, thereby reducing production costs and improving the operability and universality of the process. The process is simple and suitable for industrial large-scale production. DETAILED DESCRIPTION

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0019] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0020] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0021] Example 1 A method for preparing Fmoc-Thr(tBu)-OH, specifically: (1) Add 1500 mL of anhydrous methanol to the reaction flask, cool it to 0°C, and add dichlorothionyl (375 g, 3.15 mol, 1.5 eq) dropwise. Maintain the internal temperature at no more than 10°C during the addition. After the addition is complete, react at 0-10°C for 1 hour. Then add L-threonine (250 g, 2.1 mol), slowly raise the temperature to 30-35°C and react for 24-48 hours. After the reaction is complete, the reaction solution is concentrated to dryness under reduced pressure with hot water, and 500 mL of anhydrous ethyl methanol is added to dissolve it. Then, it is concentrated to dryness under reduced pressure to obtain threonine methyl ester hydrochloride.

[0022] (2) Add 1600 mL of anhydrous methanol to the reaction flask, stir and dissolve L-threonine methyl ester hydrochloride, cool to 0°C, and add 267 g of triethylamine (267 g, 2.6 mol) and stir for 30 minutes. Control the temperature not to exceed 5°C, add ethyl trifluoroacetate (358 g, 2.52 mol, 1.2 eq) dropwise, and slowly raise the temperature to 25°C after completion of the dropwise addition and react for 12 to 24 hours. The end point is confirmed by TLC. After the reaction is completed, the reaction solution is concentrated to dryness, and then 1600 mL of ethyl acetate is added and stirred to dissolve, filtered, and the filtrate is washed with 1N dilute hydrochloric acid, saturated sodium bicarbonate, and saturated brine in sequence, dried, filtered, and concentrated to obtain 470 g of the oily intermediate trifluoroacetyl-L-threonine methyl ester with a yield of 97.75%.

[0023] Dissolve trifluoroacetyl-L-threonine methyl ester (56 g, 0.24 mol) obtained in the previous step in 500 mL of dichloromethane, cool to 0-5°C, then add 7 g of concentrated sulfuric acid and introduce isobutylene (40 g, 0.72 mol, 3 eq). React for 12-20 hours. Monitor the complete disappearance of the reaction starting materials by a plate. Adjust the pH to 7-7.5 by adding 5% sodium bicarbonate solution at 0-5°C and allow the mixture to stand for separation.

[0024] (3) The organic phase obtained in the previous step was cooled to 10-12°C, and 2N sodium hydroxide solution (370 mL) was slowly added to the system. The saponification reaction was carried out at 10-15°C for 6 hours. The reaction was monitored by a plate. The reaction was allowed to stand for stratification and the organic phase was discarded. (4) Sodium carbonate (29.1 g, 0.275 mol) was added to the aqueous phase, stirred to dissolve, acetone (250 ml) was added, and Fmoc-OSu (76.8 g, 0.23 mol) solid was added in batches, and then the reaction was carried out at 25-35 °C for 3 hours. The Fmoc-OSu completely disappeared and a trace amount of threonine tert-butyl ether remained. 250 mL of water was added to the reaction solution for dilution, and then ethyl acetate was added for extraction. The aqueous phase was filtered, and then the temperature was lowered to 0 °C. 4N hydrochloric acid was slowly added to a pH of 2-3. Solid crystals gradually appeared in the reaction system. The reaction system was stirred evenly, filtered, and washed with water. The solid was recrystallized from ethanol-water to obtain a white solid. The solid was dried to obtain 84.8 g of fluorenylmethyloxycarbonyl-L-threonine tert-butyl ether (Fmoc-Thr(tBu)-OH) as a white solid with a yield of 89% and a purity greater than 99%.

[0025] The above description is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this field, several variations and improvements can be made without departing from the creative concept of the present invention, which all fall within the scope of protection of the present invention.

Claims

1. A method for preparing Fmoc-Thr(tBu)-OH, characterized in that: The preparation process includes four steps: S1, S2, S3, and S4. The preparation process is as follows: 。 2. The method for preparing Fmoc-Thr(tBu)-OH according to claim 1, wherein The S1 is the preparation of Tfa-Thr-Ome by methyl esterification of L-threonine. The reagents and reaction conditions are anhydrous methanol, dichlorothionine, and L-threonine. The temperature is slowly raised to 30-35° C. and the reaction is carried out for 24-48 hours.

3. The method for preparing Fmoc-Thr(tBu)-OH according to claim 1, wherein The S2 includes Tfa-Thr-OMe to prepare the intermediate trifluoroacetyl-L-threonine methyl ester, and the trifluoroacetyl-L-threonine methyl ester and isobutylene are catalyzed to prepare Tfa-Thr(tBu)-OMe. The reagents and reaction conditions for preparing the intermediate trifluoroacetyl-L-threonine methyl ester from Tfa-Thr-OMe are anhydrous methanol, Tfa-Thr-Ome, triethylamine, and ethyl trifluoroacetate, and the temperature is slowly raised to 25°C and reacted for 12 to 24 hours.

4. The method for preparing Fmoc-Thr(tBu)-OH according to claim 3, wherein The reagents and reaction conditions for the catalytic reaction of trifluoroacetyl-L-threonine methyl ester and isobutylene are trifluoroacetyl-L-threonine methyl ester, dichloromethane, concentrated sulfuric acid, isobutylene, and the reaction temperature is 0-5° C. for 12-20 hours.

5. The method for preparing Fmoc-Thr(tBu)-OH according to claim 1, wherein The S3 is Tfa-Thr(tBu)-OMe alkaline saponification and hydrolysis to prepare H-Thr(tBu)-OH.

6. The method for preparing Fmoc-Thr(tBu)-OH according to claim 1, wherein The S4 is Fmoc-protected on H-Thr(tBu)-OH to obtain Fmoc-Thr(tBu)-OH. The reagents and reaction conditions are sodium carbonate, acetone, and Fmoc-Osu, and the reaction is carried out at 25-35° C. for 3 hours.

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