A green process for the preparation of fully protected dipeptides

Abstract

The application discloses a green preparation method of a fully-protected dipeptide and belongs to the technical field of polypeptide synthesis. The method comprises the following steps: in step one, Fmoc-Thr(tBu)-OH is reacted with N-hydroxysuccinimide in an organic solvent in the presence of a condensing agent to generate an Fmoc-Thr(tBu)-OSu activated ester, wherein the N-hydroxysuccinimide is added in a batch feeding mode, the temperature is controlled at 20-25 DEG C during the feeding, the temperature is raised to 35 DEG C after the feeding is completed, and the reaction is carried out; after the reaction is completed, two times of slurry purification are carried out; in step two, the activated ester is added into a water / methanol / tetrahydrofuran mixed solvent, and a coupling reaction is carried out with glycine in the presence of an inorganic base at 20-25 DEG C; the post-treatment is carried out by extraction, acid washing, salt washing, drying, concentration and slurry, and an Fmoc-Thr(tBu)-Gly dipeptide is obtained. Through the synergistic optimization of a condensing agent, an alkali system, a solvent system and a post-treatment process, the high-yield and high-purity preparation of the fully-protected dipeptide is realized, the HPLC purity of the product can reach 99.96%, and the method is suitable for the preparation of various dipeptides such as Boc-Gly-Leu and Fmoc-Ile-Aib.

Description

Technical Field

[0001] This application relates to the field of polypeptide synthesis technology, specifically a green preparation method for a fully protected dipeptide. Background Technology

[0002] In solid-phase peptide synthesis, Fmoc-Thr(tBu)-Gly-OH is often used as a dipeptide building block for the synthesis of target peptides containing threonine-glycine sequences. Existing methods for preparing such dipeptides mainly include two technical routes:

[0003] The first approach involves condensing Boc-Thr(tBu) with glycine methyl ester hydrochloride, followed by deprotection and Fmoc introduction. However, this route is lengthy and prone to acylation of threonine side chain hydroxyl groups during condensation. Furthermore, steps such as Boc removal and methyl ester saponification result in low yields and complex byproducts, hindering large-scale preparation.

[0004] The second approach involves first converting Fmoc-Thr(tBu)-OH into an activated ester, then condensing it with glycine or its ester derivative to directly obtain Fmoc-Thr(tBu)-Gly-OH. This activated ester method has significant advantages, including fewer reaction steps, milder conditions, higher coupling efficiency, and controllable side reactions, and has become the mainstream technology for preparing this dipeptide. However, this route still faces several challenges in practical applications:

[0005] (1) The production cost of DMF, a commonly used solvent, is relatively high, and its use is accompanied by significant environmental hazards. It has a high boiling point and is difficult to recycle, which does not meet the requirements of green chemistry development.

[0006] (2) The purification process of the key intermediate activated ester requires multiple column chromatography separations, which is cumbersome, time-consuming, labor-intensive, and consumes a lot of solvent, which seriously restricts the economy and sustainability of the process.

[0007] (3) The Fmoc protecting group is prone to removal side reactions under alkaline conditions, especially under strong alkaline, high temperature or long reaction conditions, the Fmoc removal rate can reach 2-3%, which seriously affects the purity of the product;

[0008] (4) Most existing technologies are only for the preparation of single dipeptide compounds, and lack systematic research on the applicable scope of substrates and the universality of processes.

[0009] Therefore, developing a method for preparing fully protected dipeptides that has high yield, high purity, stable protecting groups, simple post-processing (no column chromatography required), is environmentally friendly, and has a wide range of applications has significant industrial application value. Summary of the Invention

[0010] To address the problems of high DMF solvent costs and significant environmental risks in existing processes, the reliance on column chromatography for activated ester purification, the easy removal of Fmoc protecting groups, and limited substrate applicability, this application provides a green method for the preparation of fully protected dipeptides. While retaining the core advantages of the activated ester method, this method achieves efficient and green preparation of high-quality fully protected dipeptides through synergistic optimization of the type of condensing agent, batch feeding temperature control, alkali system, solvent system, and post-treatment process, effectively overcoming the shortcomings of the aforementioned existing technologies.

[0011] This application is achieved through the following technical solution:

[0012] A green method for preparing a fully protected dipeptide includes the following steps:

[0013] Step 1: Fmoc-Thr(tBu)-OH and N-hydroxysuccinimide are reacted in an organic solvent in the presence of a condensing agent to generate Fmoc-Thr(tBu)-OSu activated ester. The condensing agent is selected from any one of DCC, DIC, and EDCI, and the amount of the condensing agent is 1.0~1.5 equivalents of Fmoc-Thr(tBu)-OH, and the amount of N-hydroxysuccinimide is 1.0~1.5 equivalents of Fmoc-Thr(tBu)-OH. The N-hydroxysuccinimide is added in batches, and the system temperature is controlled at 20-25℃ during the addition. After the N-hydroxysuccinimide is added, the system temperature is raised to 35℃ for reaction. After the reaction, the mixture is purified twice by pulping to obtain Fmoc-Thr(tBu)-OSu activated ester.

[0014] Step 2: Add the Fmoc-Thr(tBu)-OSu obtained in Step 1 to a mixed solvent of water, methanol, and tetrahydrofuran and start stirring. Add glycine and an inorganic base, selected from sodium hydroxide, sodium carbonate, and sodium bicarbonate. The amount of the inorganic base is 1.1 to 2.3 equivalents of Fmoc-Thr(tBu)-OSu, and the amount of glycine is 1.0 to 1.5 equivalents of Fmoc-Thr(tBu)-OSu. The inorganic base is added in batches, maintaining the reaction temperature at 20-25°C. After the reaction, the product is post-treated by ethyl acetate extraction, pH adjustment to 3-4 with hydrochloric acid, washing with saturated sodium chloride solution, drying, concentration, and petroleum ether slurrying to obtain the Fmoc-Thr(tBu)-Gly dipeptide.

[0015] Furthermore, the organic solvent in step one is tetrahydrofuran.

[0016] Furthermore, in step one, the condensing agent is EDCI, and the amount of N-hydroxysuccinimide used is 1.09 equivalents of Fmoc-Thr(tBu)-OH.

[0017] Furthermore, in step two, the inorganic base is sodium bicarbonate, and the amount of sodium bicarbonate used is 1.1 to 2.3 equivalents of Fmoc-Thr(tBu)-OSu.

[0018] Furthermore, in step two, the volume ratio of the mixed solvent of water, methanol, and tetrahydrofuran is 500:200:200.

[0019] Furthermore, the post-processing in step two does not involve column chromatography purification.

[0020] Furthermore, the condensing agent in step one is EDCI, and the inorganic base in step two is sodium bicarbonate; the HPLC purity of the Fmoc-Thr(tBu)-Gly dipeptide prepared by the method is ≥99.9%.

[0021] Furthermore, the method is also used to prepare Boc-Gly-Leu and Fmoc-Ile-Aib.

[0022] Furthermore, in step one of the preparation of Boc-Gly-Leu, Boc-Gly-OH is used instead of Fmoc-Thr(tBu)-OH as the starting material, and in step two, leucine is used instead of glycine as the coupling component.

[0023] Furthermore, in step one of the preparation of Fmoc-Ile-Aib, Fmoc-Ile-OH is used instead of Fmoc-Thr(tBu)-OH as the starting material, and in step two, α-aminoisobutyric acid is used instead of glycine as the coupling component.

[0024] The technical solution provided in this application has the following advantages compared with the prior art:

[0025] 1. From a reaction stoichiometric perspective, the amidation reaction of activated esters with amino acids requires approximately 2 equivalents of a monobasic base (1 equivalent for activating the amino group + 1 equivalent for neutralizing the byproduct N-hydroxysuccinimide). However, using a strong base (such as NaOH) at 2 equivalents leads to significant removal of the Fmoc protecting group (comparative Example 4, purity only 96.24%). This invention safely achieves a base dosage of 2.2 equivalents by using the weak base sodium bicarbonate instead of a strong base, ensuring both complete coupling reaction (yield 84.1%) and effectively avoiding Fmoc removal (purity 99.96%), thus achieving a synergistic balance between protecting group compatibility and reaction efficiency.

[0026] 2. This invention achieves high-yield and high-purity dipeptide preparation through synergistic optimization of the condensing agent, alkali system, and solvent system. Taking Fmoc-Thr(tBu)-Gly as an example, the overall yield can reach 69.0% (Example 3), and the HPLC purity is as high as 99.96%. Compared with the traditional DMF solvent system (overall yield of only 51.8%), the yield is significantly improved.

[0027] 3. This invention employs a THF-based composite solvent system, eliminating the need for fine purification via column chromatography. High-purity products can be obtained through simple post-processing methods such as concentration, recrystallization, and pulping, significantly simplifying the production process and reducing purification costs and organic solvent consumption. Both reaction steps are conventional liquid-phase synthesis operations, requiring no special equipment and offering significant advantages for industrial scale-up.

[0028] 4. This invention uses a THF / water / methanol mixed solvent to replace the traditional DMF solvent system, which significantly reduces the environmental impact and solvent cost of the process, and is in line with the development concept of green chemistry.

[0029] 5. This invention not only achieves the efficient synthesis of Fmoc-Thr(tBu)-Gly, but also successfully applies it to the preparation of Boc-Gly-Leu and Fmoc-Ile-Aib (yields of 86.5% and 83.9%, respectively, and purities of 99.94% and 99.74%, respectively). This process is adaptable to both Fmoc and Boc, two mainstream amino protecting groups, and is compatible with both conventional amino acids and non-natural Aib amino acids. It has a wide range of substrate applications and is well-suited for large-scale application. Attached Figure Description

[0030] To more clearly illustrate the technical solution of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 The process flow diagram for preparing Fmoc-Thr(tBu)-Gly according to the present invention is shown below;

[0032] Figure 2 This is a schematic diagram of the HPLC detection results of Example 1 of the present invention;

[0033] Figure 3 This is a schematic diagram of the HPLC detection results in Example 2 of the present invention;

[0034] Figure 4 This is a schematic diagram of the HPLC detection results in Example 3 of the present invention;

[0035] Figure 5This is a schematic diagram of the HPLC detection results of Comparative Example 4 of the present invention;

[0036] Figure 6 This is a schematic diagram of the HPLC detection results of Comparative Example 5 of the present invention;

[0037] Figure 7 This is a schematic diagram of the HPLC detection results of Comparative Example 6 of the present invention;

[0038] Figure 8 This is a schematic diagram of the HPLC detection results of Comparative Example 7 of the present invention;

[0039] Figure 9 This is a schematic diagram of the HPLC detection results in Example 8 of the present invention;

[0040] Figure 10 This is a schematic diagram of the HPLC detection results in Example 8 of the present invention;

[0041] Figure 11 The image shows the hydrogen NMR spectrum of Fmoc-Thr(tBu)-Gly from Example 3. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. The components of the embodiments of this application described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0043] Therefore, the following detailed description of the embodiments of this application provided in the accompanying drawings is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments of the application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0044] Example 1 (DCC+NaOH system)

[0045] Step 1: Preparation of Fmoc-Thr(tBu)-OSu

[0046] Add 1 L of tetrahydrofuran (THF) and 397 g (1.0 mol) of Fmoc-Thr(tBu) sequentially to a clean 2 L three-necked glass reaction flask. Turn on the mechanical stirrer and adjust the stirring speed to 200-300 rpm to fully dissolve the raw materials. Stir for about 15-20 min until the reaction solution becomes clear and transparent. Add 247.2 g (1.2 mol) of N,N'-dicyclohexylcarbodiimide (DCC) to the reaction flask and continue stirring for 10 min to completely dissolve the DCC.

[0047] 125g (1.09mol) of N-hydroxysuccinimide (HOSu) was divided into 5 equal batches, each approximately 25g. One batch of HOSu was added every 10 minutes while maintaining the reaction system temperature at 20-25℃, with each batch added within 3-5 minutes. During the addition process, the temperature was controlled using a circulating cooling water bath outside the reaction flask, and the internal temperature was monitored in real time using a thermometer inserted into the reaction solution to ensure the temperature remained within the 20-25℃ range. After all HOSu was added, the cooling water bath was removed, and the reaction system was heated to 35℃ and maintained at this temperature for 5 hours. Samples were taken every hour during the reaction for TLC monitoring. The TLC developing solvent was petroleum ether (PET):ethyl acetate (EA) = 1:1 (volume ratio), and observations were made under a UV lamp at 254nm. The reaction was considered complete when the Fmoc-Thr(tBu)-OH starting material spot (Rf≈0.3) disappeared.

[0048] After the reaction is complete, filter the mixture while it is still hot (about 35°C) using a Buchner funnel (medium-speed qualitative filter paper), and collect the filter cake and filtrate (mother liquor 1). Transfer the filter cake to a clean 2L beaker, add 0.5L of THF, stir and wash with a glass rod for 30 minutes, and then filter again, collecting the washing filtrate (mother liquor 2). Combine mother liquor 1 and mother liquor 2.

[0049] The combined mother liquor was transferred to a receiving flask in a 3L rotary evaporator and concentrated under reduced pressure at a water bath temperature of 40-45℃ and a vacuum degree ≤-0.08MPa. Concentration was stopped when most of the THF was distilled off and the residue in the flask became viscous (approximately 100-150mL). 100mL of ethyl acetate (EA) and 300mL of petroleum ether (PET) were added to the concentrated residue, and stirring was started (approximately 300rpm). A large amount of white solid gradually precipitated out. Stirring was continued for 30 minutes to allow the crystals to fully precipitate, and the crude product was obtained by filtration.

[0050] The crude product was transferred to a 1L single-necked flask, and approximately 300mL of THF was added. The mixture was heated to 50-55℃ and stirred to dissolve. After the solid was completely dissolved, the mixture was slowly cooled to room temperature, and then 100mL of EA and 300mL of PET were added. The mixture was stirred and crystallized for 30 minutes, followed by filtration. The recrystallization process was repeated once, for a total of two crystallization purifications. The resulting white crystalline solid was placed in a vacuum drying oven and dried under vacuum at 40℃ for 6 hours to obtain Fmoc-Thr(tBu)-OSu. The final product weighed 400g, with a yield of 80.9%.

[0051] Step 2: Preparation of Fmoc-Thr(tBu)-Gly

[0052] Using 400g (0.81mol) of Fmoc-Thr(tBu)-OSu obtained in step one as the starting material, it was added to a clean 3L three-necked glass reaction flask. 500mL of deionized water, 200mL of methanol (MeOH), and 200mL of tetrahydrofuran (THF) were added sequentially. Mechanical stirring was started, and the speed was adjusted to 200-300 rpm. Stirring was carried out for approximately 15 minutes to ensure the activated ester was fully dissolved or uniformly suspended.

[0053] 60.7 g (0.81 mol) of glycine (Gly) was added to the reaction flask and stirred for 10 min to ensure uniform dispersion of glycine in the mixed solvent. Then, 36 g (0.90 mol) of sodium hydroxide was divided into four equal batches of 9 g each, added every 15 min. During the addition process, the temperature of the reaction system was maintained at 20-25℃ using a circulating water bath. After the sodium hydroxide was added, the reaction was continued at this temperature for 4 hours. Samples were taken every hour during the reaction for TLC monitoring. The TLC developing solvent was dichloromethane (DCM):methanol (MeOH) = 5:1 (volume ratio). A dual-color development method was used: first, the UV absorption spots of the Fmoc compound were observed under a UV lamp (254 nm), and then the spots of the free amino compound were observed after heating with ninhydrin. The reaction was considered complete when the Fmoc-Thr(tBu)-OSu starting material spot (Rf≈0.6) disappeared.

[0054] After the reaction was complete, 1 L of ethyl acetate (EA) was added to the reaction system. The mixture was stirred for 5 min and then allowed to stand for approximately 15 min to allow for clear separation of the two phases. The upper organic ester layer (EA phase) was retained, and the aqueous phase was discarded. Under stirring, 3 mol / L hydrochloric acid was slowly added dropwise to the organic phase, with real-time pH monitoring using a pH meter, until the system pH was adjusted to 3-4. During the addition of hydrochloric acid, the system temperature was controlled to not exceed 25°C using an external water bath.

[0055] Transfer the acidified organic phase to a separatory funnel, allow it to stand and separate into layers, retaining the upper ester layer and discarding the lower aqueous layer. Add 300 mL of saturated sodium chloride solution to the ester layer, stir for 5 min, allow it to stand and separate into layers, retaining the upper ester layer. Repeat the washing operation with saturated sodium chloride solution once (washing twice in total). Transfer the washed ester layer to an Erlenmeyer flask, add approximately 50 g of anhydrous sodium sulfate, and dry by shaking the Erlenmeyer flask occasionally during drying. Place it at room temperature and dry overnight (approximately 12 hours).

[0056] After drying, the desiccant was removed by vacuum filtration using a Buchner funnel, and the filtrate was collected. The filtrate was transferred to a rotary evaporator and concentrated under reduced pressure at a water bath temperature of 40–45°C and a vacuum degree ≤ -0.08 MPa until the residual volume was approximately 100–150 mL (with a large amount of white solid precipitating out). While stirring (approximately 200 rpm), 300 mL of petroleum ether (PET) was slowly added dropwise to the concentrate at a rate of approximately 5 mL / min. After the addition was complete, stirring and slurrying continued for 1 hour to allow the product to fully precipitate. The product was then filtered, the white solid was collected, and dried in a vacuum drying oven at 40°C for 6 hours to obtain a white solid powder product, Fmoc-Thr(tBu)-Gly. The final product weight was 284.6 g, the yield was 77.5% (based on activated ester), and the purity was 99.71% as determined by HPLC.

[0057] This example serves as a basic scheme to verify the feasibility of preparing Fmoc-Thr(tBu)-OSu using DCC / HOSu and then coupling it with glycine in a NaOH system to prepare Fmoc-Thr(tBu)-Gly. The process involved ester activation in THF, batch addition of alkali in a water / MeOH / THF mixed solvent, temperature-controlled reaction at 20-25°C, EA extraction, acidification to pH 3-4, salt washing, drying, and EA / PET crystallization. The final yield was 400g of activated ester (80.9% yield) and 284.6g of dipeptide (77.5% yield), with an HPLC purity of 99.71%. This demonstrates the feasibility of the route and the avoidance of column chromatography in post-processing. However, given the relatively strong alkalinity of NaOH, there is still room for optimization in yield and purity.

[0058] Example 2 (DIC + Sodium Carbonate System)

[0059] Step 1: Preparation of Fmoc-Thr(tBu)-OSu

[0060] Add 1 L of tetrahydrofuran (THF) and 397 g (1.0 mol) of Fmoc-Thr(tBu) sequentially to a clean 2 L three-necked glass reaction flask. Turn on the mechanical stirrer and adjust the speed to 200-300 rpm, stirring for 15-20 min until the raw materials are completely dissolved and a clear, transparent solution is formed. Add 151.2 g (1.2 mol) of N,N'-diisopropylcarbodiimide (DIC) to the reaction flask and continue stirring for 10 min.

[0061] 125g (1.09mol) of N-hydroxysuccinimide (HOSu) was evenly divided into 5 batches, each approximately 25g. While maintaining the reaction system temperature at 20-25℃, one batch of HOSu was added every 10 minutes, with each batch added within 3-5 minutes. Temperature was controlled throughout the process using a circulating cooling water bath and monitored in real-time with a thermometer. After all HOSu was added, the cooling water bath was removed, and the system was heated to 35℃ and maintained at this temperature for 5 hours. During the reaction, samples were taken hourly for TLC monitoring using PET:EA as the developing solvent (volume ratio) at 254nm UV light. The reaction was considered complete when the Fmoc-Thr(tBu)-OH starting material spot (Rf≈0.3) disappeared.

[0062] After the reaction is complete, filter the mixture while hot using a Buchner funnel (medium-speed qualitative filter paper) and collect the filter cake and mother liquor 1. Transfer the filter cake to a 2L beaker, add 0.5L of THF, stir and wash with a glass rod for 30 minutes, then filter again and collect the washing filtrate (mother liquor 2). Combine mother liquor 1 and mother liquor 2.

[0063] The combined mother liquor was concentrated under reduced pressure in a water bath at 40-45℃ and a vacuum degree ≤-0.08MPa until the residue became viscous (approximately 100-150mL). 100mL of EA and 300mL of PET were added to the concentrated residue, and stirring was started (approximately 300rpm). A large amount of white solid precipitated out. Stirring was continued for 30 minutes, and the product was filtered to obtain the crude product. Approximately 300mL of THF was added to the crude product, and the mixture was heated to 50-55℃ to dissolve. After cooling, 100mL of EA and 300mL of PET were added for recrystallization. This recrystallization process was repeated once. The final white crystalline solid was dried in a vacuum drying oven at 40℃ for 6 hours to obtain Fmoc-Thr(tBu)-OSu. The final product weight was 410g, with a yield of 82.9%.

[0064] Step 2: Preparation of Fmoc-Thr(tBu)-Gly

[0065] Using 10g (0.83mol) of Fmoc-Thr(tBu)-OSu4 obtained in step one as the starting material, it was added to a clean 3L three-necked glass reaction flask. 500mL of deionized water, 200mL of methanol and 200mL of THF were added in sequence. Stirring was started (200~300rpm) and stirred for 15min to make the activated ester uniformly dispersed.

[0066] Add 62.2 g (0.83 mol) of glycine and stir for 10 min. Divide 105 g (0.99 mol) of anhydrous sodium carbonate into 5 equal batches, each approximately 21 g, and add one batch every 15 min. During the addition process, use a circulating water bath to control the temperature and ensure that the internal temperature of the system is stable at 20-25℃. After the addition is complete, stir the reaction at this temperature for 4 hours. Take samples hourly for TLC monitoring. The developing solvent is DCM:MeOH=5:1. TLC detection uses a dual-color method: first observe the UV absorption spots of the Fmoc compound under a UV lamp (254 nm), and then observe the spots of the free amino compound after heating with ninhydrin colorimetric reagent. The reaction is considered complete when the Fmoc-Thr(tBu)-OSu starting material spot (Rf≈0.6) disappears.

[0067] After the reaction was complete, EA1L was added to the system, stirred for 5 min, and allowed to stand for 15 min to separate the layers, retaining the upper ester layer. 3 mol / L hydrochloric acid was slowly added dropwise to the organic phase with stirring, monitoring the pH until it reached 3-4. The temperature was controlled to not exceed 25°C throughout the acidification process. The ester layer was separated, and 300 mL of saturated sodium chloride solution was added. After stirring for 5 min, the layers were allowed to separate, and the ester layer was retained. The mixture was washed once more. Approximately 50 g of anhydrous sodium sulfate was added to the ester layer, and the mixture was dried at room temperature overnight (approximately 12 h).

[0068] After drying, the desiccant was removed by filtration, and the filtrate was collected. The filtrate was then concentrated under reduced pressure at 40-45℃ in a water bath and a vacuum degree ≤-0.08MPa to a residual volume of approximately 100-150mL (a large amount of white solid precipitated). 300mL of PET was slowly added dropwise with stirring (approximately 5mL / min). After the addition was complete, the mixture was stirred for another hour. The mixture was then filtered, and the white solid was collected and dried under vacuum at 40℃ for 6 hours to obtain the white solid product Fmoc-Thr(tBu)-Gly. The final product weight was 305.6g, with a yield of 81.2% (based on activated ester), and an HPLC purity of 99.84%.

[0069] This example aims to investigate the effects of condensing agents and bases on the reaction by replacing DCC with DIC and Na2CO3 with NaOH. The operation is similar to that of Example 1, but DIC is used to prepare the activated ester and Na2CO3 is used as a weak base for coupling. The final result is 410g of activated ester with a yield of 82.9% and 305.6g of dipeptide with a yield of 81.2% and an HPLC purity of 99.84%. Compared with Example 1, the yield and purity of this system are improved, indicating that the DIC / Na2CO3 combination is feasible and that the weak base is beneficial to reducing Fmoc removal, while maintaining the advantages of simple post-processing and easy scale-up.

[0070] Example 3 (EDCI + Sodium Bicarbonate System)

[0071] Step 1: Preparation of Fmoc-Thr(tBu)-OSu

[0072] Add 1 L of tetrahydrofuran (THF) and 397 g (1.0 mol) of Fmoc-Thr(tBu) sequentially to a clean 2 L three-necked glass reaction flask. Turn on the mechanical stirrer and adjust the speed to 200-300 rpm, stirring for 15-20 min until completely dissolved and a clear solution is formed. Add 230.0 g (1.2 mol) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI·HCl) to the reaction flask, and continue stirring for 10 min to dissolve.

[0073] 125g (1.09mol) of N-hydroxysuccinimide (HOSu) was evenly divided into 5 batches of 25g each. While maintaining the reaction system temperature at 20-25℃, one batch of HOSu was added every 10 minutes, with each batch added over 3-5 minutes. Temperature was precisely controlled throughout the process using a circulating cooling water bath, and the internal temperature was monitored in real-time with a thermometer. After all the HOSu had been added, the cooling water bath was removed, and the system was heated to 35℃ and maintained at this temperature for 5 hours. Samples were taken every hour during the reaction for TLC monitoring using PET:EA = 1:1 (volume ratio) as the developing solvent, and observed under a UV lamp at 254nm. The reaction was considered complete when the Fmoc-Thr(tBu)-OH starting material spot (Rf≈0.3) completely disappeared.

[0074] After the reaction is complete, filter the mixture while it is still hot using a Buchner funnel (medium-speed qualitative filter paper) and collect the filter cake and mother liquor 1. Transfer the filter cake to a clean 2L beaker, add 0.5L of THF, stir and wash with a glass rod for 30 minutes, and filter again to collect mother liquor 2. Combine mother liquor 1 and mother liquor 2.

[0075] The combined mother liquor was transferred to a rotary evaporator and concentrated under reduced pressure in a water bath at 40-45℃ and a vacuum degree ≤-0.08MPa until the residue became viscous (approximately 100-150mL). 100mL of EA and 300mL of PET were added to the concentrate, and stirring was started (approximately 300rpm). A large amount of white solid gradually precipitated out. Stirring continued for 30 minutes, and the product was filtered to obtain the crude product. The crude product was transferred to a 1L single-necked flask, and approximately 300mL of THF was added. The mixture was heated to 50-55℃ and stirred until dissolved and clear. The mixture was allowed to cool naturally to room temperature, and 100mL of EA and 300mL of PET were added. The mixture was stirred to induce crystallization for 30 minutes and then filtered. The recrystallization process was repeated once (a total of two crystallizations). The final white crystalline solid was dried in a vacuum drying oven at 40℃ for 6 hours to obtain Fmoc-Thr(tBu)-OSu. The final product weight was 406g, with a yield of 82.1%.

[0076] Step 2: Preparation of Fmoc-Thr(tBu)-Gly

[0077] Using the Fmoc-Thr(tBu)-OSu406g (0.82mol) obtained in step one as the starting material, it was added to a clean 3L three-necked glass reaction flask. 500mL of deionized water, 200mL of methanol and 200mL of THF were added in sequence. Mechanical stirring was started at 200~300rpm and stirred for about 15min to make the activated ester uniformly dispersed in the mixed solvent.

[0078] Add 61.5 g (0.82 mol) of glycine to the reaction flask and continue stirring for 10 min to ensure uniform dispersion. Divide 151 g (1.80 mol) of sodium bicarbonate into 6 equal batches, approximately 25 g per batch, and add one batch every 15 min. During the addition process, the temperature of the reaction system is precisely controlled at 20-25℃ using a circulating cooling water bath, and the temperature is monitored in real time with a thermometer. A small amount of gas (CO2) will be released during the addition of sodium bicarbonate, so it is necessary to keep the reaction flask vented. After all the sodium bicarbonate has been added, continue stirring at 20-25℃ for 4 hours. During the reaction, samples are taken every hour for TLC monitoring. The developing solvent is DCM:MeOH = 5:1 (volume ratio). The TLC detection uses a dual-color method: first observe the UV absorption spots of the Fmoc compound under a UV lamp (254 nm), and then observe the spots of the free amino compound after heating with ninhydrin colorimetric reagent. The reaction is considered complete when the Fmoc-Thr(tBu)-OSu feedstock spot (Rf≈0.6) completely disappears.

[0079] After the reaction is complete, add 1 L of ethyl acetate (EA) to the reaction system and stir for 5 min to ensure thorough mixing. Then allow the mixture to stand for approximately 15 min to separate into two phases. Once the two phases have clearly separated, retain the upper organic ester layer (EA layer) and discard the lower aqueous phase. Under stirring, slowly add 3 mol / L hydrochloric acid dropwise to the organic phase, monitoring the pH in real time with a precision pH meter and adjusting the pH to 3-4. The addition of hydrochloric acid is exothermic, and the system temperature must be controlled to not exceed 25°C using an external water bath.

[0080] Transfer the acidified organic phase to a separatory funnel, allow it to stand and separate into layers, retaining the upper ester layer and discarding the lower aqueous layer. Add 300 mL of saturated sodium chloride solution to the ester layer, shake and wash for 5 min, then allow it to stand and separate, retaining the ester layer. Repeat the washing procedure with saturated sodium chloride solution once. Transfer the washed ester layer to an Erlenmeyer flask, add approximately 50 g of anhydrous sodium sulfate for drying, and allow it to dry overnight (approximately 12 hours) at room temperature, shaking the Erlenmeyer flask occasionally during this period.

[0081] After drying, sodium sulfate was removed by vacuum filtration using a Buchner funnel, and the filtrate was collected. The filtrate was transferred to a rotary evaporator and concentrated under reduced pressure at a water bath temperature of 40-45℃ and a vacuum degree ≤-0.08MPa until the residual volume was approximately 100-150mL, at which point a large amount of white solid had precipitated in the flask. Under stirring (approximately 200rpm), 300mL of petroleum ether (PET) was slowly added dropwise to the concentrate at a rate controlled at approximately 5mL / min. During the addition, the amount of white solid precipitated increased significantly. After the addition was complete, stirring and slurrying continued for 1 hour to ensure complete product precipitation and uniform particle size. The white solid was collected by vacuum filtration and dried in a vacuum drying oven at 40℃ for 6 hours to obtain the white solid powder product Fmoc-Thr(tBu)-Gly. The final product weight was 313.5g, the yield was 84.1% (based on activated ester), and the purity was 99.96% as determined by HPLC.

[0082] This embodiment represents the core preferred scheme, aiming to prepare the activated ester via EDCI·HCl / HOSu and couple glycine to a weak base system of NaHCO3 to obtain the highest yield and purity. The process involves batch addition of NaHCO3, temperature control at 20-25℃, TLC dual-color monitoring, EA extraction, acidification, salt washing, drying, and PET pulping and crystallization. The final activated ester yield was 82.1%, with 313.5 g of dipeptide (yield 84.1%) and HPLC purity of 99.96%. This scheme offers mild conditions, good protecting group stability, few byproducts, and eliminates the need for column chromatography, resulting in the best overall performance and best demonstrating the advantages of this application in terms of yield, purity, and scale-up applicability.

[0083] Comparative Example 4 (High concentration NaOH, investigating Fmoc removal conditions)

[0084] Step two is basically the same as step two in Example 1, except that the amount of sodium hydroxide is increased from 36g (0.90mol) to 72g (1.80mol, i.e., 2.0 equivalents relative to Fmoc-Thr(tBu)-OSu), and the reaction time is extended from 4 hours to 8 hours. Specifically, 400g (0.81mol) of Fmoc-Thr(tBu)-OSu is added to a 3L reaction flask, followed by water / MeOH / THF (500 / 200 / 200mL), 60.7g of Gly, and 72g of NaOH is added in 6 batches of 12g each, every 15 minutes. The temperature is controlled at 20-25℃, and the reaction is allowed to proceed for 8 hours. Post-processing is the same as step two in Example 1. HPLC analysis showed that the purity of the target product was 96.24%, and mass spectrometry analysis confirmed that the content of Fmoc byproducts was approximately 3%.

[0085] This comparative example aims to investigate the effect of increasing the amount of NaOH and extending the reaction time on the Fmoc protecting group. The amount of NaOH was increased from 36g to 72g, and the reaction time was extended from 4 hours to 8 hours, with the remaining operations the same as in Example 1. The results showed that the HPLC purity of the target product decreased to 96.24%, and mass spectrometry confirmed that approximately 3% of the byproducts of Fmoc were removed. This counterexample demonstrates that strong bases and high base concentrations lead to the removal of Fmoc, proving the rationality of the choice of base amount and reaction time in Example 1, and also supporting the necessity of subsequently using weak bases such as Na2CO3 and NaHCO3.

[0086] Comparative Example 5 (high-temperature reaction, investigating Fmoc removal conditions)

[0087] Step two is basically the same as step two in Example 1, except that the reaction temperature is increased from 20-25℃ to 40℃, while the reaction time remains 4 hours. Specifically, 400g (0.81mol) of Fmoc-Thr(tBu)-OSu is added to a 3L reaction flask, followed by water / MeOH / THF (500 / 200 / 200mL), 60.7g of Gly, and 36g of NaOH is added in four batches of 9g each. The reaction is carried out at 40℃ for 4 hours. Post-processing is the same as step two in Example 1. HPLC analysis showed a target product purity of 97.65%, and mass spectrometry analysis confirmed that the content of byproducts removed from Fmoc was approximately 2%.

[0088] This comparative example aims to investigate the effect of increased temperature on the stability of the Fmoc protecting group. The reaction temperature was increased from 20-25°C to 40°C, while the rest of the operation was basically the same as in Example 1. The result showed that the HPLC purity of the target product decreased to 97.65%, and about 2% of the Fmoc byproducts were removed. This counterexample shows that although high temperature may accelerate the reaction, it will promote the removal of Fmoc and side reactions, proving that the mild temperature control of 20-25°C in this scheme is not an arbitrary choice, but a key condition to ensure purity and the integrity of the protecting group.

[0089] Comparative Example 6 (Extending the reaction time to investigate Fmoc stability conditions)

[0090] Step two is essentially the same as step two in Example 3 (EDCI + sodium bicarbonate system), except that the reaction time is extended from 4 hours to 12 hours. Specifically, 0.82 mol of Fmoc-Thr(tBu)-OSu 406 g is added to a 3 L reaction flask, followed by water / MeOH / THF (500 / 200 / 200 mL), 1.5 g of Gly, and 151 g of NaHCO3 (in 6 batches). The temperature is controlled at 20-25 °C, and the reaction is carried out for 12 hours. Post-processing is the same as step two in Example 3. HPLC analysis shows that the purity of the target product is 99.85%, and the Fmoc protecting group has not been significantly removed.

[0091] This comparative example was used to verify the stability of the Fmoc protecting group in the preferred EDCI+NaHCO3 system. The reaction time of Example 3 was extended from 4 hours to 12 hours, while the other operations remained the same. The result showed that the HPLC purity of the target product still reached 99.85%, and the Fmoc protecting group was basically not removed. This result is in stark contrast to Comparative Examples 4 and 5, indicating that the weak base NaHCO3 system has a wide time window and good compatibility with the protecting group, further supporting Example 3 as the preferred solution.

[0092] Comparative Example 7 (DMF solvent system comparison)

[0093] Step 1: Preparation of Fmoc-Thr(tBu)-OSu (DMF system)

[0094] Add 1L of N,N-dimethylformamide (DMF) and 397g (1.0mol) of Fmoc-Thr(tBu) to a clean 2L three-necked glass reaction flask. Start mechanical stirring at 200-300 rpm for 15-20 minutes until completely dissolved. Add 247.2g (1.2mol) of DCC to the reaction flask and stir for 10 minutes to dissolve. Add 127g (1.10mol) of HOSu in 5 batches, maintaining the temperature at 20-25℃ during the addition process. After the addition is complete, raise the temperature to 35℃ and react for 5 hours. Monitor with TLC (PET:EA = 1:1). After the reactants have reacted completely, add 2L of water to the reaction system. A large amount of white solid precipitates. Stir for 30 minutes and then filter. Dissolve the filter cake in 1L of EA, transfer to a separatory funnel, and wash twice with 300mL of saturated sodium chloride solution, retaining the organic phase. The organic phase was dried overnight with anhydrous sodium sulfate, filtered, concentrated to a small volume, and purified by silica gel column chromatography (eluting with PET:EA = 1:2 → 1:1 gradient elution). The target eluent fraction was collected, combined, concentrated, and dried to obtain 352 g of activated ester, with a yield of 71.2% and an HPLC purity of 98.3%.

[0095] Step 2: Preparation of Fmoc-Thr(tBu)-Gly (DMF system)

[0096] Take 352 g (0.71 mol) of the activated ester obtained in step one and add it to a 3 L three-necked reaction flask. Add 500 mL of water, 200 mL of methanol, and 200 mL of DMF (using DMF instead of THF as a co-solvent) sequentially. Start stirring, add 53.3 g (0.71 mol) of Gly, and add 31.7 g (0.79 mol) of NaOH in portions. Control the temperature at 20-25℃ and react for 4 hours. Monitor the reaction by TLC (DCM:MeOH=5:1). After the reaction is complete, extract with EA1L, adjust the pH to 3-4 with 3 mol / L HCl, separate the ester layer, wash twice with saturated sodium chloride solution, and dry overnight with anhydrous sodium sulfate. After filtration and concentration, purify by silica gel column chromatography (eluting with a gradient of DCM:MeOH=10:1→5:1). Collect the target fraction, concentrate and dry to obtain 236.5 g of product. The overall yield of the two steps is 51.8%, and the HPLC purity is 99.67%.

[0097] This comparative example aims to investigate the effect of replacing THF with DMF as a co-solvent on yield and post-processing. The activated ester preparation yield was only 71.2%, with an HPLC purity of 98.3%. The dipeptide was purified by silica gel column chromatography to obtain 236.5 g, with a total two-step yield of 51.8% and an HPLC purity of 99.67%. This example shows that although the DMF system can achieve high purity, the yield is significantly low, the post-processing is complex and dependent on column chromatography, making it unsuitable for industrial scale-up. This contrasts with the advantages of the proposed solution, which uses a water / MeOH / THF mixed solvent combined with the EA / PET crystallization route, in terms of yield, ease of operation, and scale-up potential.

[0098] Example 8 (Preparation of Boc-Gly-Leu, verifying the universality of the process)

[0099] Step 1: Preparation of Boc-Gly-OSu

[0100] Add 1L of THF and 175.2g (1.0mol) of Boc-Gly-OH to a clean 2L three-necked glass reaction flask. Turn on the mechanical stirrer at 200-300 rpm and stir for 15-20 minutes until completely dissolved. Add 230.4g (1.2mol) of EDCI·HCl to the reaction flask and stir for 10 minutes to dissolve.

[0101] 138.1 g (1.2 mol) of HOSu was evenly divided into 5 batches, each approximately 27.6 g. One batch was added every 10 minutes, with each batch added over 3-5 minutes, while maintaining the system temperature at 20-25°C. A circulating cooling water bath was used for temperature control throughout the process. After all HOSu was added, the temperature was raised to 35°C and the reaction was carried out for 5 hours. TLC monitoring was performed hourly during the reaction, using PET:EA as the developing solvent (1:1). Observation was conducted using UV light and ninhydrin. The reaction was considered complete when the Boc-Gly-OH starting material spot (Rf≈0.2) disappeared.

[0102] After the reaction was complete, the mixture was filtered while hot using a Buchner funnel, and the filter cake and mother liquor 1 were collected. The filter cake was slurried and washed with 0.5 L of THF for 30 min, then filtered again, and mother liquor 2 was collected. Mother liquor 1 and mother liquor 2 were combined. The combined mother liquors were concentrated under reduced pressure in a water bath at 40–45 °C and a vacuum of ≤-0.08 MPa until the residue became viscous (approximately 100–150 mL). 100 mL of EA and 300 mL of PET were added, and the mixture was stirred for 30 min to induce crystallization. The resulting crude product was obtained by filtration. Approximately 300 mL of THF was added to the crude product, and the mixture was heated to 50–55 °C to dissolve it. The mixture was then cooled and recrystallized. This crystallization process was repeated once. The white crystalline solid was dried under vacuum at 40 °C for 6 hours to obtain 231.5 g of Boc-Gly-OSu, with a yield of 84.2%.

[0103] Step 2: Preparation of Boc-Gly-Leu

[0104] Using 31.5 g (0.842 mol) of Boc-Gly-OSu2 obtained in step one as the starting material, it was added to a clean 3L three-necked reaction flask. 500 mL of deionized water, 200 mL of methanol, and 200 mL of THF were added sequentially. Stirring was started (200-300 rpm) for 15 min to ensure uniform dispersion of the activated ester. 110.2 g (0.842 mol) of leucine (Leu) was added, and stirring was continued for 10 min. 162.2 g (1.93 mol) of sodium bicarbonate was evenly divided into 6 batches, approximately 27 g per batch, added every 15 min, maintaining the temperature at 20-25℃ during the addition process. After all the materials were added, the reaction was continued at 20-25℃ for 4 hours, with hourly TLC monitoring (developing solvent DCM:MeOH = 5:1, ninhydrin for color development). The reaction was stopped after the starting material spots disappeared.

[0105] Post-processing: Extract with EA1L, stir for 5 min, and allow to stand for 15 min to separate the layers, retaining the ester layer. Adjust the pH to 3-4 with 3 mol / L HCl (monitor with a pH meter, control temperature ≤25℃), separate the layers, and retain the ester layer. Wash the ester layer twice with 300 mL of saturated sodium chloride solution, and dry overnight (12 h) with approximately 50 g of anhydrous sodium sulfate. Remove the desiccant by suction filtration, concentrate the filtrate to approximately 100-150 mL in a water bath at 40-45℃ and a vacuum degree ≤-0.08 MPa, slowly add 300 mL of PET (approximately 5 mL / min) with stirring, slurry for 1 hour, filter, and dry under vacuum at 40℃ for 6 hours to obtain 231.8 g of white solid Boc-Gly-Leu, yield 86.5% (based on activated ester), HPLC purity 99.94%.

[0106] This example aims to verify the universality of the proposed process for Boc protecting groups and leucine substrates. Boc-Gly-OSu was prepared using EDCI / HOSu, then coupled with Leu in a weakly alkaline NaHCO3 system. Following EA extraction, acidification, salt washing, drying, and PET pulping and crystallization, the final yield of Boc-Gly-OSu was 84.2%, and the yield of Boc-Gly-Leu was 231.8 g (86.5%) with an HPLC purity of 99.94%. This example demonstrates that the proposed method is not only applicable to Fmoc systems but also compatible with Boc protecting groups and Leu substrates, and it eliminates the need for column chromatography, further proving the process's versatility and scale-up potential.

[0107] Example 9 (Preparation of Fmoc-Ile-Aib, verifying the universality of the process)

[0108] Step 1: Preparation of Fmoc-Ile-OSu

[0109] Add 1L of THF and 3.4g (1.0mol) of Fmoc-Ile-OH to a clean 2L three-necked glass reaction flask. Turn on the mechanical stirrer at 200-300 rpm and stir for 15-20 minutes until completely dissolved. Add 1.2g (1.2mol) of DIC15 to the reaction flask and stir for 10 minutes.

[0110] 138.1 g (1.2 mol) of HOSu was evenly divided into 5 batches, each approximately 27.6 g. One batch was added every 10 minutes, with each batch added over 3-5 minutes, while maintaining the system temperature at 20-25°C. The entire process was carried out using a circulating cooling water bath for temperature control. After all HOSu was added, the temperature was raised to 35°C and the reaction proceeded for 5 hours. TLC was performed hourly, with the developing solvent PET:EA = 1:1, and observation under a 254 nm UV lamp. The reaction was considered complete when the Fmoc-Ile-OH starting material spot (Rf≈0.4) disappeared.

[0111] After the reaction was complete, the mixture was filtered while hot using a Buchner funnel. The filter cake was slurried and washed with 0.5 L of THF for 30 min, then filtered again. The mother liquors were combined. The mother liquor was concentrated to a viscous consistency (approximately 100-150 mL) in a water bath at 40-45 °C under a vacuum of ≤-0.08 MPa. 100 mL of EA and 300 mL of PET were added, and the mixture was stirred for 30 min to induce crystallization. The crude product was then filtered. Approximately 300 mL of THF was added to the crude product, and the mixture was heated to 50-55 °C to dissolve it. The mixture was then cooled and recrystallized. This recrystallization process was repeated once. The white crystalline solid was dried under vacuum at 40 °C for 6 hours to obtain 365.8 g of Fmoc-Ile-OSu, with a yield of 82.7%.

[0112] Step 2: Preparation of Fmoc-Ile-Aib

[0113] Using 365.8 g (0.827 mol) of Fmoc-Ile-OSu3 obtained in step one as the starting material, it was added to a clean 3L three-necked reaction flask. 500 mL of deionized water, 200 mL of methanol, and 200 mL of THF were added sequentially, and stirring was started (200-300 rpm) for 15 min to ensure uniform dispersion of the activated ester. 85.3 g (0.827 mol) of α-aminoisobutyric acid (Aib) was added, and stirring was continued for 10 min. 175.4 g (1.65 mol) of sodium carbonate was evenly divided into 6 batches, approximately 29 g per batch, added every 15 min. The temperature was controlled at 20-25℃ using a circulating water bath during the addition process. After all the materials were added, the reaction was continued at 20-25℃ for 4 hours, with hourly TLC monitoring (developing solvent DCM:MeOH = 5:1, ninhydrin for color development). The reaction was stopped after the starting material spots disappeared.

[0114] Post-processing: Extract with EA1L, stir for 5 min, and allow to stand for 15 min to separate the layers, retaining the ester layer. Adjust the pH to 3-4 with 3 mol / L HCl (monitor with a pH meter, control temperature ≤25℃), and separate the layers, retaining the ester layer. Wash the ester layer twice with 300 mL of saturated sodium chloride solution, and dry overnight (12 h) with approximately 50 g of anhydrous sodium sulfate. Remove the desiccant by filtration, concentrate the filtrate to approximately 100-150 mL in a water bath at 40-45℃ and a vacuum degree ≤-0.08 MPa, slowly add 300 mL of PET (approximately 5 mL / min) with stirring, slurry for 1 hour, filter, and dry under vacuum at 40℃ for 6 hours to obtain 326.5 g of white solid Fmoc-Ile-Aib, yield 83.9% (based on activated ester), HPLC purity 99.74%.

[0115] This example aims to verify the applicability of the proposed process to Fmoc protecting groups and sterically hindered Ile and Aib substrates. Fmoc-Ile-OSu was prepared using DIC / HOSu, and then coupled with α-aminoisobutyric acid in a Na2CO3 system. After similar post-treatment, Fmoc-Ile-OSu yielded 82.7%, and Fmoc-Ile-Aib yielded 326.5 g (83.9%) with an HPLC purity of 99.74%. This example demonstrates that the proposed method can still achieve high yields and purity for β-branched amino acids and α,α-disubstituted amino acids, indicating a wide substrate applicability and further enhancing the universality and inventiveness of the technical solution.

[0116] Comparative analysis of condensing agent types:

[0117] Comparison dimensions: Example 1 (DCC) vs. Example 2 (DIC) vs. Example 3 (EDCI)

[0118] Comparison Projects Example 1 Example 2 Example 3 Condensing agent DCC DIC EDCI Step 1 Yield 80.9% 82.9% 82.1% Step 2 Alkali System NaOH <![CDATA[Na2CO3]]> <![CDATA[NaHCO3]]> Step 2 Yield 77.5% 81.2% 84.1% Product purity 99.71% 99.84% 99.96%

[0119] Although the three condensing agents all belong to the carbodiimide class, they exhibit significant differences in their performance in this process:

[0120] (1) DCC system (Example 1): DCC is the most classic carbodiimide condensing agent. Its byproduct, dicyclohexylurea (DCU), has extremely low solubility in organic solvents and can be removed by filtration. However, complete filtration of DCU is difficult in practice, and a small amount of residual DCU may affect the purity of subsequent coupling reactions. The yield and purity of Example 1 are the lowest among the three.

[0121] (2) DIC system (Example 2): The byproduct of DIC, diisopropylurea (DIU), has a certain solubility in organic solvents and can be removed by extraction and washing. Compared with DCC, DIC is liquid and easier to handle. The yield and purity of Example 2 are better than those of the DCC system.

[0122] (3) EDCI system (Example 3): The byproduct of EDCI, 1-ethyl-3-(3-dimethylaminopropyl)urea (EDU), has excellent water solubility and can be easily and thoroughly removed by washing with water. This characteristic makes the purification in step one more efficient and reduces the interference of impurities on the coupling reaction in step two. In addition, EDCI hydrochloride is water-soluble, and excess condensing agent and its byproducts can be removed by extraction and washing without column chromatography. The yield and purity of Example 3 are the best among the three.

[0123] Conclusion: EDCI performs best in the process of this invention due to the good water solubility of its byproducts and ease of post-treatment removal. DCC, DIC, and EDCI are not simple equivalents, and the superiority of EDCI had not been systematically proven prior to this invention.

[0124] Comparative analysis of alkaline systems:

[0125] Comparison dimensions: Example 1 (NaOH) vs. Example 2 (Na2CO3) vs. Example 3 (NaHCO3)

[0126] Comparison Projects Example 1 Example 2 Example 3 Step 2: Use alkali NaOH (strong alkali) <![CDATA[Sodium carbonate (moderately strong base)]]> <![CDATA[NaHCO3 (weak base)]]> pKa of base 15.7 10.3 (Conjugate acid) 6.4 (Conjugate Acids) Alkali dosage (equivalent) 1.1 1.2 2.2 Step 2 Yield 77.5% 81.2% 84.1% Product purity 99.71% 99.84% 99.96% Fmoc stability good good Excellent

[0127] Difference analysis:

[0128] (1) Strong base NaOH system (Example 1): Sodium hydroxide provides the strongest alkaline environment, which can efficiently promote the nucleophilic attack of the amino group of glycine on the activated ester, resulting in the fastest coupling reaction rate. However, the strong base environment poses a potential threat to the Fmoc protecting group, and it needs to be used under strict control of the amount of base and the reaction temperature. Example 1 has a purity of 99.71%, the lowest among the three.

[0129] (2) Medium-strong base Na2CO3 system (Example 2): Sodium carbonate is milder than NaOH, the pH of the reaction system is relatively controllable, and it has better compatibility with Fmoc protecting groups. The yield (81.2%) and purity (99.84%) of Example 2 are better than those of the NaOH system.

[0130] (3) Weak base NaHCO3 system (Example 3): Sodium bicarbonate has the mildest alkalinity and poses almost no risk of removing the Fmoc protecting group. Although a slightly higher amount (2.2 equivalents) is required to maintain a suitable alkaline environment, the reaction process is stable and controllable with minimal side reactions. Example 3 achieved the best yield and purity, and comparison with Example 6 confirmed that even with an extended reaction time of 12 hours, the Fmoc group remained stable (purity 99.85%).

[0131] Conclusion: The strength of the base is negatively correlated with the stability of the Fmoc protecting group. The weak base NaHCO3 is the optimal choice, maximizing the protection of the Fmoc group while ensuring coupling efficiency. The choice between sodium bicarbonate and sodium hydroxide is not a simple "strong-weak substitution," but a crucial choice that achieves a precise balance between "reactivity" and "protecting group stability."

[0132] Analysis of the impact of key process parameters on Fmoc stability:

[0133] Comparison dimensions: Comparison of Example 4 (high concentration of NaOH), Example 5 (high temperature), and Example 6 (extended reaction time) with Examples 1 / 3.

[0134] Comparison Projects Example 1 Example 3 Comparison 4 Comparison 5 Comparison 6 Types and dosages of alkali NaOH / 1.1 equivalent <![CDATA[NaHCO3 / 2.2 equivalents]]> NaOH / 2.0 equivalent NaOH / 1.1 equivalent <![CDATA[NaHCO3 / 2.2 equivalents]]> reaction temperature 20-25℃ 20-25℃ 20-25℃ 40℃ 20-25℃ reaction time 4h 4h 8h 4h 12h Product purity 99.71% 99.96% 96.24% 97.65% 99.85% Fmoc removal rate — — ~3% ~2% Basically none

[0135] Difference analysis:

[0136] (1) Alkali strength and dosage (comparison 4 vs Example 1): When the amount of NaOH increased from 1.1 equivalents to 2.0 equivalents and the reaction time was extended to 8 hours, the Fmoc removal rate was about 3% and the product purity dropped significantly to 96.24%. This shows that the Fmoc protecting group is extremely sensitive to the amount of strong alkali, and exceeding a certain threshold will trigger obvious removal side reactions.

[0137] (2) Reaction temperature (comparison 5 vs Example 1): When the reaction temperature was increased from 20-25℃ to 40℃, even if the amount of NaOH remained unchanged at 1.1 equivalents, the Fmoc removal rate was still about 2%, and the product purity dropped to 97.65%. This shows that the increase in temperature also significantly promotes the removal of Fmoc.

[0138] (3) Reaction time (comparison 6 vs Example 3): When a weak base NaHCO3 system was used and the reaction time was extended to 12 hours (3 times the conventional reaction time), the Fmoc group was basically not removed, and the product purity was still as high as 99.85%. This shows that under weakly alkaline conditions, the Fmoc protecting group has excellent time and temperature stability.

[0139] Conclusion: The stability of the Fmoc protecting group is affected by the combined effects of base strength, base dosage, reaction temperature, and reaction time. The preferred conditions of "weak base + room temperature + appropriate base dosage" in this invention solve the core problem of protecting group removal in the synthesis of Fmoc-like dipeptides from the root of the process. Strong base, high temperature, and excessive base are all key risk factors leading to Fmoc removal.

[0140] Comparative analysis of solvent systems:

[0141] Comparison Dimensions: Example 7 (DMF system) vs. Examples 1-3 (THF system)

[0142] Comparison Projects Comparative Example 7 Examples 1-3 Step 1 Solvent DMF THF Step 2 Solvent DMF / Water / Methanol THF / Water / Methanol Post-processing of step one Column chromatography purification Purification by two processes Step 2 Post-processing Column chromatography purification Extraction-washing-pulping Step 1 Yield 71.2% 80.9~82.9% Total return 51.8% 62.7~69.0% Product purity 99.67% 99.71~99.96%

[0143] Difference analysis:

[0144] (1) Fundamental difference in post-processing methods: The DMF system (comparison 7) requires silica gel column chromatography purification in both step one and step two post-processing, which is cumbersome, time-consuming, and consumes a lot of solvent, making it unsuitable for industrial scale-up. In contrast, the THF system of this invention only requires concentration and recrystallization / pulping to obtain a high-purity product, and the entire process does not require column chromatography.

[0145] (2) Significant improvement in yield: The total yield of the DMF system is only 51.8%, while the total yield of the THF system of the present invention is at least 62.7% (Example 1) and at most 69.0% (Example 3), with a yield improvement of 10.9 to 17.2 percentage points.

[0146] (3) Environmental friendliness and economy: DMF has a high boiling point (153℃), is difficult to recycle, and has reproductive toxicity, which does not meet the requirements of green chemistry. THF has a low boiling point (66℃), is easy to recycle, and can form a green mixed solvent system with water / methanol, which significantly reduces the environmental impact.

[0147] Conclusion: This invention uses THF instead of DMF as the main solvent, which not only brings about a fundamental change in the post-processing method (column-free chromatography), but also achieves a dual improvement in yield and purity.

[0148] Substrate universality verification analysis:

[0149] Comparison dimensions: Comparison of Examples 8 (Boc-Gly-Leu), 9 (Fmoc-Ile-Aib) with Examples 1-3 (Fmoc-Thr(tBu)-Gly)

[0150] Comparison Projects Examples 1-3 Example 8 Example 9 Protecting base type Fmoc Boc Fmoc Carboxyl components Thr(tBu) Gly Ile amino components Gly Leu Aib Step 1 Yield 80.9~82.9% 84.2% 82.7% Step 2 Yield 77.5~84.1% 86.5% 83.9% Total return 62.7~69.0% 72.8% 69.4% Product purity 99.71~99.96% 99.94% 99.74%

[0151] Difference analysis:

[0152] (1) Applicability of Boc protecting groups (Example 8): Boc protecting groups are relatively stable under alkaline conditions (Boc removal is usually carried out under acidic conditions), and the process of the present invention is fully applicable to them. The total yield of Boc-Gly-Leu reached 72.8% and the purity was 99.94%, further demonstrating that the process has good compatibility with different protecting group systems.

[0153] (2) Applicability of sterically hindered non-natural amino acid Aib (Example 9): Aib (α-aminoisobutyric acid) has two methyl groups, and its steric hindrance is significantly greater than that of conventional amino acids. Under conventional coupling conditions, the reaction is often slow and the yield is low. Under the process of this invention, the total yield of Fmoc-Ile-Aib still reaches 69.4% and the purity is 99.74%, proving that the process is also effective for sterically hindered substrates.

[0154] Conclusion: The process of this invention is not only applicable to the preparation of Fmoc-Thr(tBu)-Gly, but also successfully extended to Boc-Gly-Leu and Fmoc-Ile-Aib, covering two mainstream protecting groups (Fmoc / Boc) and two substrate types (conventional amino acids / non-natural amino acids). Examples 8-9 use essentially the same operating procedures as Examples 1-3, further demonstrating the robustness and versatility of this process as a platform technology, providing a scalable and universal method for dipeptide preparation in the field of peptide synthesis.

[0155] Based on the comparative analysis of the above dimensions, the core advantages of the technical solution of this invention are as follows: Based on the amino acid-activated ester route, through a synergistic design of "mild weak alkali + water / MeOH / THF mixed solvent + batch feeding with temperature control + EA / PET crystallization post-treatment," weak alkalis such as NaHCO3 and Na2CO3 are added in batches at 20-25℃, effectively suppressing Fmoc removal and side reactions, while being compatible with protecting groups such as Boc and tBu; the preferred EDCI+NaHCO3 system yields a dipeptide yield of 84.1% and HPLC purity of 99.96%, and other embodiments also maintain high yields and purity; the post-treatment involves EA extraction and acidification to pH... 3-4. Washing with saturated brine, drying with anhydrous sodium sulfate, concentration, and PET pulping and crystallization are all sufficient to complete the process, eliminating the need for silica gel column chromatography and avoiding the drawbacks of low yield, complex post-processing, and difficulty in scale-up associated with DMF systems. Furthermore, this method can be extended to different protecting groups and sterically hindered substrates such as Boc-Gly-Leu and Fmoc-Ile-Aib, demonstrating good substrate versatility. Therefore, this method simultaneously achieves the core advantages of mild conditions, stable protecting groups, high yield, high purity, simple operation, and ease of industrial scale-up.

[0156] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A green preparation method for a fully protected dipeptide, characterized in that, Includes the following steps: Step 1: Fmoc-Thr(tBu)-OH and N-hydroxysuccinimide are reacted in an organic solvent in the presence of a condensing agent to generate Fmoc-Thr(tBu)-OSu activated ester. The condensing agent is selected from any one of DCC, DIC, and EDCI, and the amount of the condensing agent is 1.0~1.5 equivalents of Fmoc-Thr(tBu)-OH, and the amount of N-hydroxysuccinimide is 1.0~1.5 equivalents of Fmoc-Thr(tBu)-OH. The N-hydroxysuccinimide is added in batches, and the system temperature is controlled at 20-25℃ during the addition. After the N-hydroxysuccinimide is added, the system temperature is raised to 35℃ for reaction. After the reaction, the mixture is purified twice by pulping to obtain Fmoc-Thr(tBu)-OSu activated ester. Step 2: Add the Fmoc-Thr(tBu)-OSu obtained in Step 1 to a mixed solvent of water, methanol, and tetrahydrofuran and start stirring. Add glycine and an inorganic base, selected from sodium hydroxide, sodium carbonate, and sodium bicarbonate. The amount of the inorganic base is 1.1 to 2.3 equivalents of Fmoc-Thr(tBu)-OSu, and the amount of glycine is 1.0 to 1.5 equivalents of Fmoc-Thr(tBu)-OSu. The inorganic base is added in batches, maintaining the reaction temperature at 20-25°C. After the reaction, the product is post-treated by ethyl acetate extraction, pH adjustment to 3-4 with hydrochloric acid, washing with saturated sodium chloride solution, drying, concentration, and petroleum ether slurrying to obtain the Fmoc-Thr(tBu)-Gly dipeptide.

2. The green preparation method of a fully protected dipeptide according to claim 1, characterized in that: The organic solvent in step one is tetrahydrofuran.

3. The green preparation method of a fully protected dipeptide according to claim 1, characterized in that: In step one, the condensing agent is EDCI, and the amount of N-hydroxysuccinimide used is 1.09 equivalents of Fmoc-Thr(tBu)-OH.

4. The green preparation method of a fully protected dipeptide according to claim 1, characterized in that: In step two, the inorganic base is sodium bicarbonate, and the amount of sodium bicarbonate used is 1.1 to 2.3 equivalents of Fmoc-Thr(tBu)-OSu.

5. The green preparation method of a fully protected dipeptide according to claim 1, characterized in that: In step two, the volume ratio of the mixed solvent of water, methanol, and tetrahydrofuran is 500:200:

200.

6. The green preparation method of a fully protected dipeptide according to claim 1, characterized in that: The post-processing in step two does not involve column chromatography purification.

7. The green preparation method of a fully protected dipeptide according to claim 1, characterized in that: The condensing agent in step one is EDCI, and the inorganic base in step two is sodium bicarbonate; the HPLC purity of the Fmoc-Thr(tBu)-Gly dipeptide prepared by the method is ≥99.9%.

8. A green preparation method for a fully protected dipeptide according to any one of claims 1-7, characterized in that: The method is also used to prepare Boc-Gly-Leu or Fmoc-Ile-Aib.

9. The green preparation method of a fully protected dipeptide according to claim 8, characterized in that: In step one of the preparation of Boc-Gly-Leu, Boc-Gly-OH is used instead of Fmoc-Thr(tBu)-OH as the starting material, and in step two, leucine is used instead of glycine as the coupling component.

10. The green preparation method of a fully protected dipeptide according to claim 8, characterized in that: In step one of the preparation of Fmoc-Ile-Aib, Fmoc-Ile-OH is used instead of Fmoc-Thr(tBu)-OH as the starting material, and in step two, α-aminoisobutyric acid is used instead of glycine as the coupling component.

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