A method for synthesizing Fmoc-NH-PEG3-CH2COOH (Fmoc-AEEEA)

Abstract

This invention relates to the field of organic synthesis, specifically to a method for synthesizing Fmoc-NH-PEG3-CH2COOH. This method avoids the use of highly toxic and explosive azides, features mild reaction conditions, is simple to operate, and produces high-yield and high-purity products, significantly reducing production costs and making it suitable for industrial production, thus alleviating the economic burden on patients.

Description

Technical Field

[0001] This invention relates to the field of organic synthesis, and more specifically to a method for synthesizing Fmoc-NH-PEG3-CH2COOH. Background Technology

[0002] Fmoc-NH-PEG3-CH2COOH is a bifunctional molecule containing an amino group protected by a fluorenemethyloxycarbonyl (Fmoc) group, a polyethylene glycol (PEG3) segment, and a carboxymethyl (-CH2COOH) group. This compound is commonly used as a key intermediate or structural unit in peptide synthesis, medicinal chemistry, and bioconjugation.

[0003] In solid-phase peptide synthesis, Fmoc-NH-PEG3-CH2COOH can serve as a module for introducing PEG segments, increasing the hydrophilicity and flexibility of peptides or peptide-mimicking drugs and improving their bioactivity. In the development of antibody-drug conjugates (ADCs) and protein degradation-targeting chimeric compounds (PROTACs), this compound can serve as a linker precursor. Its terminal carboxyl group can be used to couple with amino groups on antibody or target protein ligands, while the Fmoc-protected amino group can react with other functional groups (such as carboxyl groups on drug molecules or E3 ligase ligands) after deprotection. The PEG segment can effectively increase the water solubility of the linker, reduce aggregation tendency, and optimize the pharmacokinetic properties of the entire molecule. Furthermore, this compound is often used as a starting material in the preparation of functionalized polyethylene glycol materials for further modification and construction of complex biomaterials.

[0004] Currently, methods for synthesizing Fmoc-NH-PEG3-CH2COOH typically involve multiple and hazardous reactions. For example, they may begin with ethylene glycol and highly toxic and explosive azides, proceeding through azidation-reduction, carboxylation, amino protection, and the introduction of Fmoc groups. Each step requires purification using column chromatography and other cumbersome methods, which are not only time-consuming and labor-intensive but also result in low yields and high costs, generating large quantities of organic solutions. Some routes may also use hazardous or expensive reagents, hindering industrial-scale production.

[0005] Therefore, developing a synthetic method for Fmoc-NH-PEG3-CH2COOH that is mild in reaction conditions, simple in operation, low in cost, and suitable for large-scale production is of great significance for reducing the research and development and production costs of related drugs and materials, and benefiting patients. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, this invention provides an efficient and simple method for synthesizing Fmoc-NH-PEG3-CH2COOH, which solves the problems of complex synthesis routes, high risk, low yield, and high cost.

[0007] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: This invention provides a method for synthesizing Fmoc-NH-PEG3-CH2COOH, the method comprising the following steps: Step 1: Diethylene glycol is reacted with an organic base catalyst in an organic solvent, and then sodium chloroacetate is added. After post-treatment and column chromatography separation, intermediate a is obtained. Step 2: Dissolve intermediate a in an organic solvent, add bromoacetonitrile, and carry out a substitution reaction under controlled temperature under sodium hydride conditions. After treatment, intermediate b is obtained. Step 3: Intermediate b is reduced by sodium borohydride and then post-processed to obtain intermediate c; Step 4: Intermediate c is reacted with Fmoc-OSu under alkaline conditions to introduce the Fmoc protecting group. After acidification and extraction, the target product Fmoc-NH-PEG3-CH2COOH is obtained.

[0008] Preferably, the organic solvent used in the first step of synthesizing intermediate a is one or more of tetrahydrofuran, N,N-dimethylformamide, and acetonitrile. Step 1 yields both monosubstituted (i.e., intermediate a) and disubstituted products.

[0009] Preferably, the organic base catalyst used in the first step of synthesizing intermediate a is one of potassium tert-butoxide (t-BuOK) or sodium ethoxide (EtONa), and its amount is 1.0 to 1.2 times the molar amount of diethylene glycol.

[0010] Preferably, the molar amount of diethylene glycol is 3 to 5 times the molar amount of sodium chloroacetate.

[0011] Preferably, in the first step, the reaction temperature for diethylene glycol to generate diethylene glycol anions with the organic base catalyst is 0~10℃, and the reaction time is 30~60 minutes.

[0012] Preferably, the nucleophilic substitution reaction between the diethylene glycol oxonium and sodium chloroacetate is carried out at a temperature of 25-35°C and for a reaction time of 5-9 hours.

[0013] Preferably, the reaction process is monitored by thin-layer chromatography or high-performance liquid chromatography (HPLC) to control the amount of disubstituted byproducts to be less than 25%.

[0014] Preferably, the crude product is purified by silica gel column chromatography (using a gradient elution system of dichloromethane / methanol).

[0015] Preferably, the reaction temperature in the second step is 0~10℃ and the reaction time is 9-16 hours.

[0016] Optionally, in the second step of synthesizing intermediate b, the amount of sodium hydride used is 3-5 times the molar amount of intermediate a.

[0017] Preferably, the organic solvent used in the second step reaction is one or more of anhydrous tetrahydrofuran and dioxane.

[0018] Preferably, the amount of bromoacetonitrile used is 1.5-2.5 times the molar amount of intermediate a.

[0019] Preferably, the reaction temperature in the third step is 0-10℃, and the reaction time is 15-20 hours.

[0020] Preferably, the amount of sodium borohydride used is 3-5 times the molar amount of intermediate b.

[0021] Preferably, the reagent used to introduce the Fmoc protecting group in the fourth step is Fmoc-OSu, and its amount is 0.98 to 1.1 times the molar amount of intermediate c.

[0022] Preferably, the fourth step reaction is carried out under alkaline conditions, and the alkali used is either sodium bicarbonate or sodium carbonate.

[0023] Preferably, the organic solvent used in the fourth step reaction is one or more of tetrahydrofuran, dioxane, and acetonitrile, which together with water form a mixed solvent system.

[0024] Preferably, the reaction temperature in the fourth step is 17~25℃ and the reaction time is 4~7 hours.

[0025] Preferably, the reaction formula is: In this invention, if there is a conflict between the Chinese name and the structural formula of a compound, the structural formula shall prevail, unless the structural formula is obviously incorrect.

[0026] The beneficial effects of this invention are as follows: the synthesis method provided by this invention avoids the use of highly toxic and explosive azides, the reaction conditions are mild, the operation is simple, the product yield is high and the purity is high, the production cost is significantly reduced, it is suitable for industrial production, and it helps to alleviate the economic burden on patients. Attached Figure Description

[0027] Figure 1 The nuclear magnetic resonance spectrum of Fmoc-NH-PEG3-CH2COOH obtained in Specific Example 2 of this application; Figure 2 The high-performance liquid chromatogram of Fmoc-NH-PEG3-CH2COOH obtained in Specific Example 2 of this application; Figure 3This is the mass spectrum of Fmoc-NH-PEG3-CH2COOH obtained in Specific Example 2 of this application. Detailed Implementation

[0028] The present invention is illustrated below with reference to examples, but is not intended to limit the invention. Any simple substitutions or modifications made to the present invention by those skilled in the art are within the scope of the technical solutions protected by this invention.

[0029] The preparation method of Fmoc-NH-PEG3-CH2COOH in this invention is mainly achieved through the following steps: Diethylene glycol and an organic solvent were added to a three-necked reaction flask equipped with a magnetic stir bar. An organic base catalyst was added in portions under ice bath conditions. After stirring, sodium chloroacetate was added, and the mixture was heated to room temperature. After the reaction was complete, ice water was poured in, the pH was adjusted to 2 with hydrochloric acid, the mixture was extracted with ethyl acetate, washed with saturated brine, dried, concentrated, and purified by column chromatography to obtain intermediate a.

[0030] Sodium hydride and an organic solvent were added to another three-necked flask, which was then cooled in an ice bath. The organic solvent solution of intermediate a was slowly added, followed by the dropwise addition of an organic solvent solution of bromoacetonitrile (temperature controlled ≤10℃). After the addition was complete, the reaction was stirred at room temperature. The reaction was quenched with methanol, filtered, and concentrated. The residue was dissolved in ethyl acetate, washed successively with dilute hydrochloric acid and saturated brine, dried, and concentrated to obtain intermediate b.

[0031] Intermediate b was dissolved in anhydrous methanol, and sodium borohydride was added in portions under ice bath conditions. The mixture was then allowed to naturally warm to room temperature. After cooling again, concentrated hydrochloric acid was slowly added dropwise until the pH reached approximately 2. The methanol was removed by concentration, and the solution was dissolved in water. The solution was washed with ethyl acetate, and the aqueous phase was alkalized with sodium carbonate solution. The solution was extracted with dichloromethane, dried, and concentrated to obtain intermediate c.

[0032] Intermediate C was dissolved in an organic solvent / water mixture, and alkali was added. Fmoc-OSu was added in portions at 17-25°C, and the reaction was stirred for 4-7 hours. The pH of the reaction solution was adjusted to 3-4 with dilute hydrochloric acid, extracted with ethyl acetate, washed with saturated brine, dried, and concentrated to obtain the target product Fmoc-NH-PEG3-CH2COOH.

[0033] The above scheme will be described in detail below under different reaction conditions: Example 1:

[0034] Diethylene glycol (63.6 g, 0.6 mol) and 100 mL of anhydrous tetrahydrofuran were added to a three-necked flask. Under nitrogen protection, potassium tert-butoxide (67.3 g, 0.6 mol) was added in portions, maintaining the temperature between 0 and 5 °C. After the addition was complete, the mixture was stirred for one hour, and then sodium chloroacetate (23.3 g, 0.2 mol) was slowly added. The temperature was raised to 25 °C, and the mixture was stirred for 9 hours. The mixture was then poured into ice water, and 3 M hydrochloric acid was added dropwise until the pH stabilized at 2. The mixture was extracted with ethyl acetate, washed with saturated brine, dried, and concentrated. Column chromatography was used to obtain intermediate a 15.92 g. The reaction was monitored by TLC, with Rf = 0.3. The yield was 48.51% based on sodium chloroacetate (evolving solvent: n-butanol: water: acetic acid = 4:1:1).

[0035] Weighed sodium hydride (11.67 g, 0.2910 mol) and 80 mL anhydrous THF were added to a three-necked flask. The mixture was placed in an ice-water bath, stirred, and cooled to 0–5 °C. Intermediate a (15.92 g, 0.0970 mol) and 40 mL anhydrous THF were slowly added to the NaH suspension. Bromoacetonitrile (17.41 g, 0.1455 mol) was mixed with 30 mL anhydrous THF and slowly added dropwise through a dropping funnel to the vigorously stirred reaction mixture, strictly controlling the temperature not to exceed 10 °C. After the addition was complete, the ice bath was removed, and the reaction mixture was allowed to return to room temperature naturally. The mixture was stirred at room temperature for 16 hours. After the reaction was completed, methanol (10 mL) was slowly added dropwise to quench the reaction until no obvious bubbles were produced. The reaction mixture was filtered under reduced pressure and concentrated to obtain a viscous, oily crude product. The crude product was dissolved in 50 mL of ethyl acetate and transferred to a separatory funnel. The solution was washed successively with pre-cooled 1M dilute hydrochloric acid aqueous solution (20 mL × 2) and saturated saline solution (20 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated again under reduced pressure to give 15.75 g of intermediate b, a pale yellow to colorless transparent oil, with a yield of 84.35%.

[0036] In a three-necked flask under nitrogen protection and ice-water bath cooling, intermediate b (15.75 g, 0.0818 mol) was dissolved in 150 mL of anhydrous methanol, maintaining the temperature at 0–5 °C. Sodium borohydride (8.79 g, 0.2325 mol) was slowly added in four batches with vigorous stirring for 30 minutes. The reaction mixture was then allowed to warm naturally to room temperature and stirred at room temperature for 20 hours. The reaction mixture was then cooled again to 0 °C. Under good stirring and cooling, 0.3 equivalents of hydrochloric acid were slowly added dropwise to quench the reaction and adjust the pH to approximately 2.0. The mixture was concentrated under reduced pressure to remove most of the methanol. The viscous residue was dissolved in 100 mL of water, washed with ethyl acetate, and the aqueous phase was alkalized with sodium carbonate solution to pH 6.5–7.5, followed by extraction with dichloromethane. The combined organic phases were dried, filtered, and concentrated to give 11.83 g of a pale yellow oily product, intermediate c, with an actual yield of 91.25%.

[0037] In a three-necked flask, intermediate c (15.54 g, 0.0746 mol), sodium bicarbonate (12.53 g, 0.1492 mol), and 60 mL of dioxane / water (1:1) mixed solvent were added. Fmoc-OSu (25.71 g, 0.07459 mol) was added in portions at 17 °C, and the mixture was stirred for 4 hours after the addition was complete. The reaction was monitored for completeness. The pH of the reaction solution was adjusted to 3-4 with dilute hydrochloric acid, 60 mL of water was added, and the mixture was extracted with ethyl acetate (3 × 60 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 26.04 g of the pale yellow, bright oily target product Fmoc-NH-PEG3-CH2COOH (a waxy solid at low temperature), with a yield of 81.3% and a purity of 98.33% (HPLC). The overall yield was 30.35%. Example 2:

[0038] Diethylene glycol (63.6 g, 1.0 mol) and 100 mL of anhydrous tetrahydrofuran were added to a three-necked flask. Under nitrogen protection, the temperature was controlled between 0 and 5 °C. Potassium tert-butoxide (67.3 g, 1.2 mol) was added in portions. After the addition was complete, the mixture was stirred for one hour. Then, sodium chloroacetate (23.3 g, 0.2 mol) was slowly added. The temperature was raised to 25 °C and stirred for 9 hours. The mixture was poured into ice water, and 3 M hydrochloric acid was added dropwise until the pH stabilized at 2. The mixture was extracted with ethyl acetate, washed with saturated brine, dried, and concentrated. Column chromatography was used to obtain intermediate a 16.43 g. The reaction was monitored by TLC. Rf = 0.3. The yield was calculated to be 50.01% based on sodium chloroacetate (evolving solvent: n-butanol: water: acetic acid = 4:1:1).

[0039] Weigh out 20.02 g (0.5000 mol) of sodium hydride and 80 mL of anhydrous THF in a three-necked flask. Place the mixture in an ice-water bath, stir, and cool to 0–5 °C. Slowly add intermediate a (16.43 g (0.1000 mol)) and 40 mL of anhydrous THF to the NaH suspension. Mix bromoacetonitrile (29.99 g (0.2500 mol) with 30 mL of anhydrous THF and slowly add the mixture dropwise through a dropping funnel to the vigorously stirred reaction mixture, strictly controlling the temperature not to exceed 10 °C. After the addition is complete, remove the ice bath and allow the reaction mixture to return to room temperature naturally. Stir the reaction mixture at room temperature for 16 hours. After the reaction is complete, slowly add 20 mL of methanol to quench the reaction until no obvious bubbles are produced. Filter the reaction mixture under reduced pressure and concentrate to obtain a viscous, oily crude product. The crude product was dissolved in 50 mL of ethyl acetate and transferred to a separatory funnel. It was then washed successively with pre-cooled 1M dilute hydrochloric acid aqueous solution (20 mL × 2) and saturated saline solution (20 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated again under reduced pressure to give 17.92 g of intermediate b, a pale yellow to colorless transparent oil, with a yield of 88.2%.

[0040] In a three-necked flask under nitrogen protection and ice-water bath cooling, intermediate b (17.92 g, 0.08822 mol) was dissolved in 150 mL of anhydrous methanol, maintaining the temperature at 0–5 °C. Sodium borohydride (16.70 g, 0.4412 mol) was slowly added in four batches with vigorous stirring for 30 minutes. The reaction mixture was then allowed to warm naturally to room temperature and stirred at room temperature for 20 hours. The reaction mixture was then cooled back to 0 °C. Under good stirring and cooling, 0.3 equivalents of hydrochloric acid were slowly added dropwise to quench the reaction and adjust the pH to approximately 2.0. The mixture was concentrated under reduced pressure to remove most of the methanol. The viscous residue was dissolved in 100 mL of water, washed with ethyl acetate, and the aqueous phase was alkalized with sodium carbonate solution to pH 6.5–7.5, followed by extraction with dichloromethane. The combined organic phases were dried, filtered, and concentrated to give 17 g of a pale yellow oily product, intermediate c, with an actual yield of 92.45%.

[0041] In a three-necked flask, intermediate c (17 g, 0.08156 mol), sodium bicarbonate (13.69 g, 0.1631 mol), and 30 mL of tetrahydrofuran / water (1:1) mixed solvent were added. Fmoc-OSu (27.53 g, 0.08156 mol) was added in portions at 19 °C, and the mixture was stirred for 5 hours after the addition was complete. The reaction was monitored for completeness. The pH of the reaction solution was adjusted to 3-4 with dilute hydrochloric acid, 30 mL of water was added, and the mixture was extracted with ethyl acetate (3 × 30 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 28.93 g of the pale yellow, bright oily target product Fmoc-NH-PEG3-CH2COOH (a waxy solid at low temperature), with a yield of 82.7% and a purity of 99.13% (HPLC). The overall yield was 33.72%. Example 3:

[0042] Diethylene glycol (63.6 g, 1.0 mol) and 100 mL of anhydrous tetrahydrofuran were added to a three-necked flask. Under nitrogen protection, the temperature was controlled between 0 and 5 °C. Potassium tert-butoxide (67.3 g, 1.2 mol) was added in portions. After the addition was complete, the mixture was stirred for one hour. Then, sodium chloroacetate (23.3 g, 0.2 mol) was slowly added. The temperature was raised to 25 °C and stirred for 5 hours. The mixture was poured into ice water, and 3 M hydrochloric acid was added dropwise until the pH stabilized at 2. The mixture was extracted with ethyl acetate, washed with saturated brine, dried, and concentrated. Column chromatography was used to obtain intermediate a 15.60 g. The reaction was monitored by TLC. Rf = 0.3. The yield was 47.52% based on sodium chloroacetate (evolving solvent: n-butanol: water: acetic acid = 4:1:1).

[0043] Weighed sodium hydride (19.04 g, 0.4752 mol) and 80 mL anhydrous THF were added to a three-necked flask. The mixture was placed in an ice-water bath, stirred, and cooled to 0–5 °C. Intermediate a (15.6 g, 0.09504 mol) and 40 mL anhydrous THF were slowly added to the NaH suspension. Bromoacetonitrile (28.51 g, 0.2376 mol) was mixed with 30 mL anhydrous THF and slowly added dropwise through a dropping funnel to the vigorously stirred reaction mixture, strictly controlling the temperature not to exceed 10 °C. After the addition was complete, the ice bath was removed, and the reaction mixture was allowed to return to room temperature naturally. The mixture was stirred at room temperature for 9 hours. After the reaction was completed, methanol (20 mL) was slowly added dropwise to quench the reaction until no obvious bubbles were produced. The reaction mixture was filtered under reduced pressure and concentrated to obtain a viscous, oily crude product. The crude product was dissolved in 50 mL of ethyl acetate and transferred to a separatory funnel. The solution was washed successively with pre-cooled 1M dilute hydrochloric acid aqueous solution (20 mL × 2) and saturated saline solution (20 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated again under reduced pressure to give 17.02 g of intermediate b, a pale yellow to colorless transparent oil, with a yield of 88.13%.

[0044] In a three-necked flask under nitrogen protection and ice-water bath cooling, intermediate b (17.02 g, 0.08376 mol) was dissolved in 150 mL of anhydrous methanol, maintaining the temperature at 0–5 °C. Sodium borohydride (12.68 g, 0.3351 mol) was slowly added in four portions with vigorous stirring for 30 minutes. The reaction mixture was then allowed to warm naturally to room temperature and stirred at room temperature for 20 hours. The reaction mixture was then cooled back to 0 °C. Under good stirring and cooling, 0.3 equivalents of hydrochloric acid were slowly added dropwise to quench the reaction and adjust the pH to approximately 2.0. The mixture was concentrated under reduced pressure to remove most of the methanol. The viscous residue was dissolved in 100 mL of water, washed with ethyl acetate, and the aqueous phase was alkalized with sodium carbonate solution to pH 6.5–7.5, followed by extraction with dichloromethane. The combined organic phases were dried, filtered, and concentrated to give 16.03 g of a pale yellow oily product, intermediate c, with an actual yield of 91.87%.

[0045] Intermediate C (16.03 g, 0.07695 mol), sodium bicarbonate (12.92 g, 0.1539 mol), and 60 mL of acetonitrile / water (1:1) mixed solvent were added to a three-necked flask. Fmoc-OSu (25.97 g, 0.0769 mol) was added in portions at 19 °C, and the mixture was stirred for 6 hours after the addition was complete. The reaction was monitored for completeness. The pH of the reaction solution was adjusted to 3-4 with dilute hydrochloric acid, 60 mL of water was added, and the mixture was extracted with ethyl acetate (3 × 60 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 27.76 g of the pale yellow, bright oily target product Fmoc-NH-PEG3-CH2COOH (a waxy solid at low temperature), with a yield of 84.1% and a purity of 98.07% (HPLC). The overall yield was 32.36%. Example 4:

[0046] Diethylene glycol (63.6 g, 1.0 mol) and 100 mL of anhydrous tetrahydrofuran were added to a three-necked flask. Under nitrogen protection, sodium ethoxide (81.7 g, 1.2 mol) was added in portions, with the temperature controlled between 0 and 5 °C. After the addition was complete, the mixture was stirred for one hour, and then sodium chloroacetate (23.3 g, 0.2 mol) was slowly added. The temperature was raised to 25 °C, and the mixture was stirred for 9 hours. The mixture was then poured into ice water, and 3 M hydrochloric acid was added dropwise until the pH stabilized at 2. The mixture was extracted with ethyl acetate, washed with saturated brine, dried, and concentrated. Column chromatography was used to obtain intermediate a 15.12 g. The reaction was monitored by TLC, with Rf = 0.3. The yield was calculated to be 46.05% based on sodium chloroacetate (evolving solvent: n-butanol: water: acetic acid = 4:1:1).

[0047] Weighed sodium hydride (18.45 g, 0.4643 mol) and 80 mL anhydrous THF were added to a three-necked flask. The mixture was placed in an ice-water bath, stirred, and cooled to 0–5 °C. Intermediate a (15.12 g, 0.09213 mol) and 40 mL anhydrous THF were slowly added to the NaH suspension. Bromoacetonitrile (27.63 g, 0.2303 mol) was mixed with 30 mL anhydrous THF and slowly added dropwise through a dropping funnel to the vigorously stirred reaction mixture, strictly controlling the temperature not to exceed 10 °C. After the addition was complete, the ice bath was removed, and the reaction mixture was allowed to return to room temperature naturally. The mixture was stirred at room temperature for 12 hours. After the reaction was completed, methanol (20 mL) was slowly added dropwise to quench the reaction until no obvious bubbles were generated. The reaction mixture was filtered under reduced pressure and concentrated to obtain a viscous, oily crude product. The crude product was dissolved in 50 mL of ethyl acetate and transferred to a separatory funnel. The solution was washed successively with pre-cooled 1M dilute hydrochloric acid aqueous solution (20 mL × 2) and saturated saline solution (20 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated again under reduced pressure to give 16.34 g of intermediate b, a pale yellow to colorless transparent oil, with a yield of 87.31%.

[0048] In a three-necked flask under nitrogen protection and ice-water bath cooling, intermediate b (16.34 g, 0.0804 mol) was dissolved in 150 mL of anhydrous methanol, maintaining the temperature at 0–5 °C. Sodium borohydride (15.22 g, 0.4021 mol) was slowly added in four portions with vigorous stirring for 30 minutes. The reaction mixture was then allowed to warm naturally to room temperature and stirred at room temperature for 15 hours. The reaction mixture was then cooled back to 0 °C. Under good stirring and cooling, 0.3 equivalents of hydrochloric acid were slowly added dropwise to quench the reaction and adjust the pH to approximately 2.0. The mixture was concentrated under reduced pressure to remove most of the methanol. The viscous residue was dissolved in 100 mL of water, washed with ethyl acetate, and the aqueous phase was alkalized with sodium carbonate solution to pH 6.5–7.5, followed by extraction with dichloromethane. The combined organic phases were dried, filtered, and concentrated to give 15.32 g of a pale yellow oily product, intermediate c, with an actual yield of 91.43%.

[0049] In a three-necked flask, intermediate c (15.32 g, 0.0735 mol), sodium bicarbonate (12.35 g, 0.1470 mol), and 60 mL of tetrahydrofuran / water (1:1) mixed solvent were added. Fmoc-OSu (27.29 g, 0.0808 mol) was added in portions at 25 °C, and the mixture was stirred for 7 hours after the addition was complete. The reaction was monitored for completeness. The pH of the reaction solution was adjusted to 3-4 with dilute hydrochloric acid, 60 mL of water was added, and the mixture was extracted with ethyl acetate (3 × 60 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 26.34 g of the pale yellow, bright oily target product Fmoc-NH-PEG3-CH2COOH (a waxy solid at low temperature), with a yield of 83.5% and a purity of 98.78% (HPLC). The overall yield was 30.70%. Example 5:

[0050] Diethylene glycol (63.6 g, 1.0 mol) and anhydrous acetonitrile were added to a three-necked flask. Under nitrogen protection, the temperature was controlled between 0 and 10 °C. Potassium tert-butoxide (67.3 g, 1.2 mol) was added in portions. After the addition was complete, the mixture was stirred for 30 minutes. Then, sodium chloroacetate (23.3 g, 0.2 mol) was slowly added. The temperature was raised to 25 °C and stirred for 9 hours. The mixture was poured into ice water, and 3M hydrochloric acid was added dropwise until the pH stabilized at 2. The mixture was extracted with dichloromethane, washed with saturated brine, dried, and concentrated. Column chromatography was used to obtain intermediate a 16.07 g. The reaction was monitored by TLC. Rf = 0.3. The yield was 48.95% based on sodium chloroacetate (evolving solvent: n-butanol: water: acetic acid = 4:1:1).

[0051] Weighed sodium hydride (19.61 g, 0.4896 mol) and 80 mL of dioxane were added to a three-necked flask. The mixture was placed in an ice-water bath, stirred, and cooled to 0–5 °C. Intermediate a (16.07 g, 0.979 mol) and 40 mL of dioxane were slowly added to the NaH suspension. Bromoacetonitrile (29.37 g, 0.2448 mol) was mixed with 30 mL of dioxane and slowly added dropwise through a dropping funnel to the vigorously stirred reaction mixture, strictly controlling the temperature not to exceed 10 °C. After the addition was complete, the ice bath was removed, and the reaction mixture was allowed to return to room temperature naturally. The mixture was stirred at room temperature for 16 hours. After the reaction was completed, methanol (20 mL) was slowly added dropwise to quench the reaction until no obvious bubbles were produced. The reaction mixture was filtered under reduced pressure and concentrated to obtain a viscous, oily crude product. The crude product was dissolved in 50 mL of ethyl acetate and transferred to a separatory funnel. It was washed successively with pre-cooled 1M dilute hydrochloric acid aqueous solution (20 mL × 2) and saturated saline solution (20 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated again under reduced pressure to give 17.19 g of intermediate b, a pale yellow to colorless transparent oil, with a yield of 86.43%.

[0052] In a three-necked flask under nitrogen protection and ice-water bath cooling, intermediate b (17.19 g, 0.08461 mol) was dissolved in 150 mL of anhydrous methanol, maintaining the temperature at 0–5 °C. Sodium borohydride (16.01 g, 0.4231 mol) was slowly added in four batches with vigorous stirring for 30 minutes. The reaction mixture was then allowed to warm naturally to room temperature and stirred at room temperature for 17 hours. The reaction mixture was then cooled back to 0 °C. Under good stirring and cooling, 0.3 equivalents of hydrochloric acid were slowly added dropwise to quench the reaction and adjust the pH to approximately 2.0. The mixture was concentrated under reduced pressure to remove most of the methanol. The viscous residue was dissolved in 100 mL of water, washed with ethyl acetate, and the aqueous phase was alkalized with sodium carbonate solution to pH 6.5–7.5, followed by extraction with dichloromethane. The combined organic phases were dried, filtered, and concentrated to give a pale yellow oily product g (intermediate c 16.10 g), with an actual yield of 91.37%.

[0053] Intermediate C (16.10 g, 0.0773 mol), sodium carbonate (16.40 g, 0.1546 mol), and 60 mL of tetrahydrofuran / water (1:1) mixed solvent were added to a three-necked flask. Fmoc-OSu (25.58 g, 0.0757 mol) was added in portions at 25 °C, and the mixture was stirred for 5 hours after the addition was complete. The reaction was monitored for completeness. The pH of the reaction solution was adjusted to 3-4 with dilute hydrochloric acid, 60 mL of water was added, and the mixture was extracted with ethyl acetate (3 × 60 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 26.61 g of the pale yellow, bright oily target product Fmoc-NH-PEG3-CH2COOH (a waxy solid at low temperature), with a yield of 80.23% and a purity of 98.12% (HPLC). The overall yield was 31.01%.

[0054] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A method of synthesizing Fmoc-NH-PEG3-CH2COOH, characterized in that, The method includes the following steps: Step 1: Diethylene glycol is reacted with an organic base catalyst in an organic solvent, and then sodium chloroacetate is added. After post-treatment and column chromatography separation, intermediate a is obtained. Step 2: Dissolve intermediate a in an organic solvent, add bromoacetonitrile, and carry out a substitution reaction under controlled temperature under sodium hydride conditions. After treatment, intermediate b is obtained. Step 3: Intermediate b is reduced by sodium borohydride and then post-processed to obtain intermediate c; Step 4: Intermediate c is reacted with Fmoc-OSu under alkaline conditions to introduce the Fmoc protecting group. After acidification and extraction, the target product Fmoc-NH-PEG3-CH2COOH is obtained.

2. The method of claim 1, wherein, The organic base catalyst in step 1 is potassium tert-butoxide or sodium ethoxide, and its amount is 1.0 to 1.2 times the molar amount of diethylene glycol.

3. The method of claim 1, wherein, In step 1, the molar amount of diethylene glycol is 3 to 5 times the molar amount of sodium chloroacetate, and the organic solvent used is one or more of tetrahydrofuran, N,N-dimethylformamide, and acetonitrile.

4. The method of claim 1, wherein, In step 1, the reaction temperature for diethylene glycol to generate diethylene glycol anions with an organic base catalyst is 0~10℃, and the reaction time is 30~60 minutes; then, the reaction temperature for adding sodium chloroacetate is 25~35℃, and the reaction time is 5~9 hours.

5. The method of claim 1, wherein, The reaction temperature in step 2 is 0~10℃, and the reaction time is 9-16 hours.

6. The method of claim 1, wherein, The amount of sodium hydride used in step 2 is 3-5 times the molar amount of intermediate a.

7. The method of claim 1, wherein, The organic solvent used in step 2 is one or more of anhydrous tetrahydrofuran and dioxane.

8. The method of claim 1, wherein, The amount of bromoacetonitrile used in step 2 is 1.5-2.5 times the molar amount of intermediate a.

9. The method of claim 1, wherein, The amount of sodium borohydride used in step 3 is 3-5 times the molar amount of intermediate b.

10. The method of claim 1, wherein, In step 4, the amount of Fmoc-OSu used is 0.98 to 1.1 times the molar amount of intermediate c, and the base used is one of sodium bicarbonate and sodium carbonate; the organic solvent used in the reaction is one or more of tetrahydrofuran, dioxane, and acetonitrile.

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