Preparation method of high-purity Fmoc-S-trityl-L-penicillamine
By dehydrating L-penicillamine with methylboric acid to form a cyclic structure, the thiol group is gently protected. Then, it is reacted with Fmoc-OSu in sodium bicarbonate buffer solution and recrystallized from ethyl acetate/n-heptane. This method solves the problem of efficient synthesis of Fmoc-S-triphenylmethyl-L-penicillamine, and obtains a high-purity and stable peptide drug intermediate suitable for industrial production.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHONGKE QUANTONG (DALIAN) PHARM TECH CO LTD
- Filing Date
- 2026-02-27
- Publication Date
- 2026-05-29
AI Technical Summary
The synthesis of Fmoc-S-triphenylmethyl-L-penicillamine in the existing technology suffers from problems such as inefficient thiol protection, high risk of racemization, difficulty in purification, and poor stability, making it difficult to meet the production needs of high-end peptide drugs.
L-Penicillamine was dehydrated with methylboric acid to form a cyclic structure. The thiol group was protected under mild conditions. Then, it was reacted with Fmoc-OSu. Racemization was inhibited using sodium bicarbonate buffer solution. Finally, it was purified by recrystallization from ethyl acetate/n-heptane to obtain a high-purity product.
It achieves high selectivity protection, low racemization and high efficiency purification, with a chemical purity of ≥99.0% and an optical purity of over 99.0%, making it suitable for large-scale production and exhibiting good product stability.
Smart Images
Figure CN122102977A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of peptide chemistry in organic synthesis, specifically relating to a method for synthesizing high-purity Fmoc-S-triphenylmethyl-L-penicillamine (abbreviated as Fmoc-L-Pen(Trt)-OH). Background Technology
[0002] Fmoc-S-triphenylmethyl-L-penicillamine is a key protected amino acid derivative used in the Fmoc solid-phase peptide synthesis (SPPS) strategy to introduce penicillamine (Pen) residues with thiol groups. Penicillamine residues play an important role in peptide and protein structures, often stabilizing higher-order structures of peptides or proteins by forming disulfide bonds (e.g., insulin, cone snail toxins). For example, WO2023288019A2 discloses Fmoc-S-triphenylmethyl-L-penicillamine as a key structural fragment for the preparation of interleukin-23 receptor inhibitors. Simultaneously, penicillamine residues can also serve as metal ion chelating sites, possessing significant application value in drug molecule design.
[0003] In existing technologies, the conventional synthetic route for Fmoc-S-triphenylmethyl-L-penicillamine typically uses L-penicillamine as the starting material, and its synthesis mainly faces the following challenges: 1. Highly efficient and specific protection of the thiol group: When the TEA / TrtCl system is used directly, triphenylmethyl (Trt-) is more likely to undergo a substitution reaction with the amino group, generating a thiol-attached byproduct. This causes the target intermediate (thiol-attached by thiol) to become a minor product. The high proportion of byproducts and low yield of the main product make subsequent separation and purification difficult, increasing production costs and failing to meet the needs of industrial production.
[0004] 2. Racemization risk: In the Fmoc group introduction step (usually using Fmoc-Cl or Fmoc-OSu), especially under alkaline conditions, there is a certain risk of racemization of the α-chiral center of penicillamine, which affects the optical purity of the final peptide product.
[0005] 3. Difficulty in purification: Crude products often contain unreacted raw materials, racemic byproducts, solvent and reagent residues, as well as impurities generated from the decomposition of unstable intermediates. Traditional purification methods (such as single recrystallization) often fail to yield high-purity products that meet the requirements of advanced peptide synthesis.
[0006] 4. Stability issues: The Trt protecting group is relatively unstable under acidic conditions. If the acidity is not properly controlled during synthesis and post-processing, it can easily lead to partial deprotection, affecting product quality and storage stability.
[0007] Therefore, developing a synthetic process that is mild, highly selective, can effectively inhibit racemization, and is easy to purify to obtain high-purity products is of great significance for ensuring the quality of penicillamine-based peptide drug research and development and production. Summary of the Invention
[0008] To address the problems existing in the prior art, this invention provides a method for preparing high-purity Fmoc-S-triphenylmethyl-L-penicillamine. This method uses L-penicillamine as a starting material, which undergoes a dehydration reaction with methylboric acid to generate a cyclic structure. Then, under mild conditions, the triphenylmethyl group selectively protects the thiol group, followed by acid addition for ring-opening to obtain the S-triphenylmethyl-L-penicillamine intermediate. Next, in a sodium bicarbonate-buffered, water / dioxane weakly alkaline system, it reacts with Fmoc-OSu to protect the amino group, effectively inhibiting racemization. Finally, it is purified by recrystallization using an ethyl acetate / n-heptane system to obtain the high-purity final product. This invention features a simple and efficient process route, mild conditions, low racemization, and a unique and effective purification method. The obtained product exhibits high chemical and optical purity, good stability, and is suitable for large-scale production, meeting the needs of high-end peptide drug synthesis.
[0009] This invention proposes and discloses a method for preparing Fmoc-S-triphenylmethyl-L-penicillamine, comprising the following three steps: Step A): L-Penicillamine is dehydrated with methylboric acid in a dioxane / toluene mixed solvent to generate a cyclic structure. Then, in the presence of an organic base, it is selectively protected with triphenylchloromethane in an organic solvent to protect the thiol group. After the reaction is completed, acid treatment is added to obtain S-triphenylmethyl-L-penicillamine. Step B): S-triphenylmethyl-L-penicillamine was reacted with 9-fluorenemethoxycarbonylsuccinimide in a mixed solvent of water and 1,4-dioxane in the presence of sodium bicarbonate, and acidified to obtain crude Fmoc-S-triphenylmethyl-L-penicillamine. Step C): Dissolve crude Fmoc-S-triphenylmethyl-L-penicillamine in a good solvent, and recrystallize by adding a poor solvent to obtain high-purity Fmoc-S-triphenylmethyl-L-penicillamine.
[0010] The reaction route is shown below:
[0011] Further, the molar ratio of triphenylchloromethane to L-penicillamine in step A) is 1.05-1.15:1, preferably 1.1:1.
[0012] Further, the organic base mentioned in step A) is selected from triethylamine, N,N-diisopropylethylamine, etc., with triethylamine being preferred.
[0013] Further, in step B), the volume ratio of water to 1,4-dioxane is 1:3-5, preferably 1:4.
[0014] Further, in step B), the molar ratio of sodium bicarbonate to S-triphenylmethyl-L-penicillamine is 2.0-3.5:1, preferably a multiple of 3.0 for sodium bicarbonate.
[0015] Further, after the reaction described in step B) is completed, the acidification treatment uses dilute acid to adjust the pH to 2-4 to precipitate the product, preferably the pH range of the precipitated solid is 2.5-3.
[0016] Further, the recrystallization described in step C) employs a combination of a good solvent and a bad solvent, wherein the good solvent is selected from at least one of dichloromethane and ethyl acetate, and the bad solvent is selected from at least one of n-heptane and petroleum ether. Preferably, the good solvent is ethyl acetate, and the bad solvent is n-heptane. The recrystallization uses a mixed solvent of ethyl acetate and cold n-heptane, with a volume ratio of 1:3-6.
[0017] Further, in step C), the solvent used for washing the crystals is a mixture of n-heptane and ethyl acetate, with a volume ratio of 3-5:1, preferably 4:1. Beneficial effects of the invention
[0018] 1. Highly selective protection: The first step involves the cyclization reaction of L-penicillamine with methylboronic acid to protect the amino group. Then, in a mixed solvent, the thiol group is efficiently and selectively protected by Trt. After acidification, the ring is opened to obtain the target intermediate, which prepares the material for the next step of the reaction.
[0019] 2. Effective suppression of racemization: The second step uses Fmoc-OSu as the acylation reagent, which has moderate activity and mild reaction conditions, and the reaction is carried out in a weakly alkaline buffer solution of sodium bicarbonate / dioxane system. The pH of this system is stably maintained at 7-8, avoiding significant racemization that may be caused by strong bases, and preserving the optical purity of the L-configuration to the greatest extent.
[0020] 3. Efficient and environmentally friendly purification process: Compared with traditional systems such as methanol / water and dichloromethane / petroleum ether, ethyl acetate has good solubility for the product, and n-heptane, as an antisolvent, has low toxicity, a moderate boiling point, and is easy to remove. This combination can effectively remove polar and non-polar impurities, including trace amounts of racemic byproducts (D-type impurities), producing crystals with good morphology, high purity, and readily acceptable residual solvent levels.
[0021] 4. Excellent product quality: The Fmoc-L-Pen(Trt)-OH prepared by this method typically has a chemical purity of ≥99.0% (area normalization method) as analyzed by high performance liquid chromatography (HPLC), and the racemic content (detected by chiral HPLC) is less than 0.3%. The product exhibits good stability when stored in a dry environment at room temperature.
[0022] 5. Stable process, suitable for scale-up: The entire process route is simple, the post-processing of each step is simple, the selected reagents are all conventional chemical raw materials, the cost is controllable, the reaction conditions are mild and safe, and it is easy to carry out laboratory-scale production from gram to kilogram levels. Attached image description: Figure 1 The image shows the high-performance liquid chromatography (HPLC) chromatogram of the final product obtained in Example 5. Figure 2 The NMR spectrum of Fmoc-S-triphenylmethyl-L-penicillamine prepared according to a specific embodiment of the present invention. Detailed Implementation
[0023] The present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.
[0024] Example 1:
[0025] In a 500 mL four-necked round-bottom flask under nitrogen protection, L-penicillamine (10.00 g, 67.1 mmol) was added, followed by 80 mL of a mixed solvent of dioxane and toluene (volume ratio 1 / 1). The mixture was stirred until the L-penicillamine was completely dissolved. Then, methylboric acid (5.20 g, 87.2 mmol) was added and stirred for 5 minutes to mix thoroughly. The four-necked flask was connected to a water separator and a reflux condenser, and the mixture was heated to 85–95 °C and refluxed to separate the water. The reaction was carried out for 6 hours until no obvious water separation occurred. Heating was then stopped, and the mixture was cooled to room temperature to obtain the cyclic protected intermediate reaction solution.
[0026] Take a small amount of the crude cyclic protecting intermediate reaction solution, dissolve it in a small amount of dichloromethane, mix with silica gel, and then evaporate to dryness. Load the mixed silica gel into the top of a pre-packed rapid silica gel column. Elute with a dichloromethane / methanol gradient and monitor by TLC. Collect the eluent corresponding to the product spot, combine them, and evaporate to dryness to obtain the pure cyclic protecting intermediate. 1 H NMR (400 MHz, DMSO-d6): 3.57 (s, 1H), 1.81 (s, 1H), 1.53(s, 1H), 1.49 (d, J = 10.2 Hz, 6H), 0.90 (s, 3H). Example 2:
[0027] In a 500 mL four-necked round-bottom flask under nitrogen protection, the intermediate reaction solution obtained in Example 1 was added to a mixed solvent of anhydrous dichloromethane (150 mL) and anhydrous DMF (15 mL). The mixture was cooled to 0–5 °C in an ice-water bath, and triethylamine (6.84 g, 67.1 mmol) and triphenylchloromethane (TrtCl) (20.58 g, 73.8 mmol) were added dropwise with stirring. After the addition was complete, the ice bath was removed, and the mixture was stirred at 20–25 °C for 10 hours. HPLC (derivative analysis) showed that the remaining L-penicillamine starting material was less than 0.1%. The reaction solution was adjusted to pH 2.5 with 1 M HCl aqueous solution and stirred for 1 hour. The organic phase was then washed with saturated brine (50 mL × 2) and dried over anhydrous sodium sulfate. The filtrate was evaporated under reduced pressure at 45–50 °C to remove the solvent, yielding approximately 25 g of a pale yellow oil. The solution was dissolved in ethyl acetate (25 mL), and then n-heptane (125 mL) was slowly added with stirring, resulting in the precipitation of a white solid. The solution was filtered, washed with n-heptane, and dried under vacuum at 50 °C to give 23.38 g of L-Pen(Trt)-OH white solid, with a yield of 89%, which was used directly in the next step.
[0028] Comparative Example 1:
[0029] To compare the effects of the method of this invention with existing methods, a comparative example was set up for the synthesis of the target intermediate L-Pen(Trt)-OH. The specific steps are as follows: The same molar amount of L-penicillamine as in Example 1 of this invention was taken and dissolved in a mixed solvent of dioxane and toluene (solvent type and amount were the same as in the example of this invention). Methylboric acid was not added; the mixture was stirred until the L-penicillamine was completely dissolved. Following the conditions of step 2 of this invention, the same molar amount of TEA was added under ice bath cooling at 0-5°C, followed by the same molar amount of TrtCl. After the addition was complete, the mixture was brought back to room temperature and stirred for 10 hours. HPLC (derivative) showed that the remaining L-penicillamine raw material was less than 0.1%. After the reaction was completed, the mixture was acidified, extracted, washed, dried, and distilled under reduced pressure according to the post-processing method of Example 2 of this invention, and recrystallized to obtain a product mixture.
[0030] The products obtained by the method of the present invention (Example) and the method of Comparative Example 1 were detected by high performance liquid chromatography (HPLC). The proportions of the main product (target intermediate, with Trt attached to the thiol group) and the by-product (with Trt attached to the amino group) and the yield of the main product were compared. The results are as follows: 1. Example 2: The proportion of main product (target intermediate) is ≥96%, the proportion of by-product (amino group attached to Trt) is ≤1%, the yield of main product is ≥89%, the product purity is high, and the subsequent separation and purification is easy.
[0031] 2. Comparative Example 1: The proportion of main product (target intermediate) is ≤30%, the proportion of by-product (amino group attached to Trt) is ≥65%, the yield of main product is ≤25%, the by-product dominates the product mixture, the main product is a minor product, and a small amount of high-purity main product can only be obtained through complex separation and purification processes, and the yield is extremely low, which cannot meet the needs of industrial production.
[0032] Example 3:
[0033] The product from the previous step, L-Pen(Trt)-OH (10 g, 25.5 mmol), was added to a mixed solvent of water (30 mL) and 1,4-dioxane (90 mL), and the mixture was cooled to 0°C in an ice bath. Sodium bicarbonate solid (6.44 g, 76.6 mmol) was added in three portions. Separately, Fmoc-OSu (9.5 g, 28.0 mmol) was dissolved in 1,4-dioxane (60 mL) and slowly added dropwise to the above reaction solution over 30 minutes. After the addition was complete, the ice bath was removed, and the mixture was stirred at 20-25°C for 6 hours. HPLC showed that less than 0.1% of the starting material remained. While cooling in an ice bath, 1M citric acid aqueous solution was slowly added dropwise to adjust the pH to 2-2.5, resulting in the precipitation of a large amount of white solid. The filter cake was filtered, washed thoroughly with cold water (20 mL × 3), and dried under vacuum at 50 °C to obtain 12.54 g of crude Fmoc-L-Pen(Trt)-OH, with a yield of 80%.
[0034] Example 4:
[0035] Intermediate L-Pen(Trt)-OH (20 g, 51.1 mmol) was added to a mixed solvent of water (60 mL) and 1,4-dioxane (120 mL), and the mixture was cooled to 0°C in an ice bath. Sodium bicarbonate solid (6.44 g, 153.3 mmol) was added in three portions. Separately, Fmoc-OSu (18.95 g, 56.2 mmol) was dissolved in 1,4-dioxane (120 mL) and slowly added dropwise to the above reaction solution over 30 minutes. After the addition was complete, the ice bath was removed, and the mixture was stirred at 20-25°C for 6 hours. HPLC showed that less than 0.1% of the starting material remained. While cooling in an ice bath, 1M citric acid aqueous solution was slowly added dropwise to adjust the pH to 2.5-3, resulting in the precipitation of a large amount of white solid. The filter cake was filtered, washed thoroughly with cold water (40 mL × 3), and dried under vacuum at 50 °C to obtain 26.65 g of crude Fmoc-L-Pen(Trt)-OH, with a yield of 85%.
[0036] Example 5:
[0037] The crude product (20 g) from Example 4 was placed in a 250 mL round-bottom flask, and ethyl acetate (100 mL) was added. The mixture was heated to 50°C and stirred to dissolve, yielding a clear solution (if there was a small amount of insoluble matter, it was filtered while hot). The hot solution was transferred to a 500 mL crystallization flask and stirred at 20-25°C. Heptane (400 mL) was then slowly added dropwise. The mixture was cooled to 0-5°C and stirred for 5 hours. After filtration, the solid was washed with a cold heptane / ethyl acetate (4 / 1) mixed solvent (40 mL) and dried in a vacuum drying oven at 50°C for 12 hours to obtain 18.1 g of a white crystalline powder, Fmoc-L-Pen(Trt)-OH, with a chemical purity of 99.4%, an optical purity of 99.8%, and a yield of 90.5%. The overall yield of the two steps was 76.9% (calculated as L-Pen(Trt)-OH).
[0038] 1 H NMR (400 MHz, DMSO-d6): δ 12.73 (s, 1H), 7.88 (d, J = 7.5 Hz, 2H), 7.78 (t, J = 6.4 Hz, 2H), 7.63 (d, J = 9.0 Hz, 1H), 7.51 (d, J = 7.7 Hz, 6H), 7.40 (t, J = 7.4 Hz, 2H), 7.32 – 7.16 (m, 12H), 4.35 – 4.17 (m, 3H), 3.90 (d, J = 9.0 Hz, 1H), 0.94 (d, J = 15.8 Hz, 6H). 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 for preparing high-purity Fmoc-S-triphenylmethyl-L-penicillamine, characterized in that, Includes the following steps: ； Step A): L-Penicillamine is dehydrated with methylboric acid in a dioxane / toluene mixed solvent to generate a cyclic structure. Then, in the presence of an organic base, it is selectively protected with triphenylchloromethane in an organic solvent to protect the thiol group. After the reaction is completed, acid treatment is added to obtain S-triphenylmethyl-L-penicillamine. Step B): S-triphenylmethyl-L-penicillamine was reacted with 9-fluorenemethoxycarbonylsuccinimide in a mixed solvent of water and 1,4-dioxane in the presence of sodium bicarbonate, and acidified to obtain crude Fmoc-S-triphenylmethyl-L-penicillamine. Step C): Dissolve crude Fmoc-S-triphenylmethyl-L-penicillamine in a good solvent, and recrystallize by adding a poor solvent to obtain high-purity Fmoc-S-triphenylmethyl-L-penicillamine.
2. The method for preparing high-purity Fmoc-S-triphenylmethyl-L-penicillamine according to claim 1, characterized in that: In step A, the molar ratio of L-penicillamine to triphenylchloromethane is 1:1.05~1.
15.
3. The method for preparing high-purity Fmoc-S-triphenylmethyl-L-penicillamine according to claim 1, characterized in that: In step A, the organic base is selected from triethylamine or N,N-diisopropylethylamine.
4. The method for preparing high-purity Fmoc-S-triphenylmethyl-L-penicillamine according to claim 1, characterized in that: In step B, the volume ratio of water to 1,4-dioxane is 1:3-5.
5. The method for preparing high-purity Fmoc-S-triphenylmethyl-L-penicillamine according to claim 1, characterized in that: In step B, the molar ratio of sodium bicarbonate to S-triphenylmethyl-L-penicillamine is 2.5-3.5:
1.
6. The method for preparing high-purity Fmoc-S-triphenylmethyl-L-penicillamine according to claim 1, characterized in that: In step B, the acidification process uses dilute acid to adjust the pH to 2-4 to precipitate the product.
7. The method for preparing high-purity Fmoc-S-triphenylmethyl-L-penicillamine according to claim 1, characterized in that: In step C, the good solvent is selected from at least one of dichloromethane and ethyl acetate, and the bad solvent is selected from at least one of n-heptane and petroleum ether.
8. The method for preparing high-purity Fmoc-S-triphenylmethyl-L-penicillamine according to claim 1, characterized in that: In step C, the good solvent is ethyl acetate, the bad solvent is n-heptane, and recrystallization uses a mixed solvent of ethyl acetate and cold n-heptane with a volume ratio of 1:3-6.