A highly efficient synthesis method of ascorbyl tripeptide-1

Abstract

This invention discloses an efficient synthetic method for succinyl ascorbic acid tripeptide-1. Starting with L-ascorbic acid, it first reacts with succinic anhydride to generate succinyl ascorbic acid, which then reacts with N-hydroxysuccinimide in the presence of a condensing agent to form a stable active ester (compound I). Simultaneously, a tripeptide-1 (Gly-His-Lys) derivative (compound II) with side chains and a C-terminus protected by a protecting group and an N-terminus free is prepared using liquid-phase or solid-phase synthesis. Compound I and compound II are coupled in an aprotic solvent in the presence of an organic base to obtain a protected intermediate. Finally, all protecting groups are removed under mild acidic conditions, and the product is purified to obtain the target product. This invention, through an active ester strategy and optimized protecting group combination, significantly improves coupling efficiency and product purity, effectively inhibits ascorbic acid degradation and peptide racemization, and has advantages such as mild reaction conditions, simple operation, high overall yield, and ease of large-scale production. It can be used to prepare high-purity succinyl ascorbic acid tripeptide-1.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide chemical synthesis, specifically relating to an efficient method for synthesizing succinyl ascorbic acid tripeptide-1. Background Technology

[0002] Ascorbic acid (vitamin C) is an important antioxidant and skin-whitening ingredient, but it suffers from poor stability and low transdermal absorption. Conjugating it with bioactive peptides (such as tripeptide-1, a copper-binding peptide that promotes collagen synthesis) can improve stability, transdermal absorption, and achieve synergistic effects.

[0003] In the prior art, the synthesis of succinylated ascorbic acid tripeptide-1 usually follows this route: first, the protected tripeptide-1 (usually with the sequence Gly-His-Lys or its derivatives) is synthesized, then it is coupled with succinylated ascorbic acid, and finally the protecting group is removed. The main problems with the existing methods are: (1) Low yield. Due to the unstable structure of ascorbic acid, side reactions such as oxidation and degradation are prone to occur in multi-step reactions, resulting in an unsatisfactory final yield. (2) Difficult purification. The crude product often contains unreacted raw materials, racemization byproducts and various protecting group byproducts, and the separation and purification steps are cumbersome and costly. (3) Harsh reaction conditions. Some steps require the use of strong acids, strong bases or high temperatures, which may destroy the active structure of ascorbic acid or peptides and increase the risk of racemization. (4) Lengthy route. Traditional solid-phase or liquid-phase synthesis methods have many steps and are complex to operate, making them unsuitable for large-scale production. Therefore, there is an urgent need to develop a high-efficiency, highly selective, mild, easy-to-purify and scale-up synthetic method. Summary of the Invention

[0004] This invention aims to overcome the shortcomings of existing technologies and provide a highly efficient method for the synthesis of succinyl ascorbic acid tripeptide-1. This method significantly improves reaction efficiency and product purity by optimizing the reaction route, selecting specific condensation reagents, combining protecting groups, and adjusting reaction conditions.

[0005] The technical solution adopted in this invention is as follows: Succinyl ascorbic acid tripeptide-1, chemical formula shown in Formula 1 below:

[0006] Formula 1 A method for synthesizing succinyl ascorbic acid tripeptide-1 includes the following steps: (1) L-ascorbic acid is reacted with succinic anhydride to generate succinyl ascorbic acid, which is then reacted with N-hydroxysuccinimide in the presence of a condensing agent to obtain succinyl ascorbic acid active ester (compound I). The structural formula of compound 1 is shown below: ; (2) Prepare a tripeptide-1 derivative (compound II) with a side chain and C-terminus protected by a protecting group and an N-terminus of a free amino group, wherein the sequence of the tripeptide-1 is Gly-His-Lys; (3) In the presence of an organic base, compound I obtained in step (1) and compound II obtained in step (2) are coupled in an aprotic solvent to obtain a protected succinyl ascorbic acid tripeptide-1 intermediate. (4) Remove all protecting groups from the intermediate obtained in step (3) and purify to obtain succinyl ascorbic acid tripeptide-1.

[0007] Step 1) involves the following steps in preparing the N-terminally protected succinyl ascorbic acid active ester: S1. L-Ascorbic acid is reacted with succinic anhydride in an aprotic solvent at low temperature (0-5°C) in the presence of a basic catalyst. The reaction solution is slowly raised to room temperature (20-25°C) and the reaction is stirred for 3-5 hours. The reaction is monitored by TLC until it is complete, and succinyl ascorbic acid intermediate is obtained. S2. The above intermediate is reacted with hydroxysuccinimide (NHS) or other active esterifying agents (such as HOBt, HBTU, etc.) under the action of condensing agents (such as DCC, EDC) to generate a stable active ester (compound I).

[0008] In S1, the aprotic solvent is DMF or DCM; the basic catalyst is at least one of triethylamine and DMAP. The molar ratio of L-ascorbic acid, succinic anhydride, and basic catalyst is 1:1.05-1.1:2.05-2.6; Preferably, the alkaline catalyst is a mixture of triethylamine and DMAP in a molar ratio of 20 to 50:1.

[0009] Based on the molar amount of L-ascorbic acid, add succinic anhydride (1.05-1.1 times the molar amount of L-ascorbic acid), triethylamine (2.0-2.5 times the molar amount of L-ascorbic acid), and DMAP (0.05-0.1 times the molar amount of L-ascorbic acid) in sequence.

[0010] The reaction temperature is 20-25°C.

[0011] In step S2, the reaction system was first cooled again to 0-5°C. Based on the molar amount of succinyl ascorbic acid generated in step S1, N-hydroxysuccinimide (NHS, with a molar amount 1.0-1.1 times that of succinyl ascorbic acid) and condensing agent EDC·HCl (with a molar amount 1.0-1.1 times that of succinyl ascorbic acid) were added sequentially. After stirring at low temperature for 1 hour, the mixture was raised to room temperature and reacted for 8-12 hours. After the reaction was completed, the mixture was extracted, washed, dried, concentrated, and purified to obtain compound I as a white solid.

[0012] In S2, the condensing agent is one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and dicyclohexylcarbodiimide (DCC); In step 2), a C-terminal and side-chain protected tripeptide-1 derivative is prepared: A tripeptide-1 derivative (compound II) was prepared by employing the Fmoc solid-phase synthesis strategy or the liquid-phase fragment condensation method. The N-terminus of the derivative was an amino group, and the imidazole nitrogen at the C-terminus and histidine (His) and the ε-amino group of the derivative (Lys) were both protected by suitable protecting groups (such as Boc, Fmoc, Trt, Pbf, etc.). Liquid-phase preparation was preferred, yielding compound II in solution or solid form.

[0013] The protecting group is independently selected from: an amino protecting group of fluorenyl methoxycarbonyl (Fmoc) or tert-butyl methoxycarbonyl (Boc); a protecting group of the imidazole nitrogen in the histidine side chain of triphenylmethyl (Trt) or 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf); a protecting group of the ε-amino group in the lysine side chain of tert-butyl methoxycarbonyl (Boc) or fluorenyl methoxycarbonyl (Fmoc); and a carboxyl protecting group of tert-butyl ester (OtBu) or benzyl ester (OBn). The preferred structure of compound II is H-Gly-His(Trt)-Lys(Boc)-OtBu or H-Gly-His(Pbf)-Lys(Fmoc)-OtBu.

[0014] In step 3), the coupling reaction is as follows: Compound I and compound II were mixed in an aprotic solvent (such as DMF, DCM, THF, or mixtures thereof) under an inert atmosphere (such as N2 or Ar) at low temperatures to room temperature (-10°C to 25°C) and reacted in the presence of an organic base (such as DIPEA or NMM). The reaction progress was monitored by TLC or HPLC, and the reaction was terminated after compound II was substantially consumed.

[0015] The molar ratio of compound I to compound II is 1.0 to 1.1:1.

[0016] In step 4), the protecting groups are removed and the product is purified. The protected target compound intermediate obtained in step 3) was reacted under mild acid deprotection conditions at low temperature (0°C) to room temperature to remove all protecting groups.

[0017] The deprotection process uses a mixture of trifluoroacetic acid (TFA) and a cleaning agent; The cleaning agent is one or more of water, triisopropylsilane (TIS), anisole, and 1,2-ethylenedithiol.

[0018] The volume fraction of TFA in the mixture is 90-97%, and the total volume fraction of the cleaning agent is 3-10%.

[0019] The deprotection mixture is preferably a mixture of trifluoroacetic acid (TFA), water, and triisopropylsilane (TIS) in a volume ratio of 95:2.5:2.5, or a mixture of TFA, water, and 1,2-ethylenedithiol (EDT) in a volume ratio of 94:3:3 (v / v / v).

[0020] After the reaction was completed, most of the TFA was removed by vacuum distillation, and the residue was added to cold diethyl ether to precipitate the crude product. The crude product was purified by high performance liquid chromatography (HPLC) or preparative chromatography, and then lyophilized to obtain high-purity succinyl ascorbic acid tripeptide-1.

[0021] The purification was performed using reversed-phase high-performance liquid chromatography with gradient elution using acetonitrile-water solution containing 0.1% trifluoroacetic acid as the mobile phase.

[0022] Succinyl ascorbic acid tripeptide-1 is a target molecule with specific instabilities. The enediol structure of ascorbic acid is extremely sensitive to oxidation, alkalis, high temperatures, metal ions, and strong electrophiles (such as carbocations), making it highly susceptible to degradation. The imidazole ring of histidine (His) readily races during peptide synthesis, and during deprotection in strong acids (such as with Boc protection), the resulting tert-butyl cation (tBu) is removed. + This will attack the imidazole ring, leading to complex alkylation byproducts. When both are combined in the final deprotection step, if tBu is generated during the removal of the His protecting group... + This highly reactive cation attacks both the imidazole ring of His and the sensitive site of ascorbic acid, causing "double damage" and resulting in low yield and a large number of byproducts.

[0023] This invention addresses the complex technical challenge of extremely low yield caused by the superposition of "ascorbic acid degradation" and "His side reaction" in the later stages of synthesis (especially the deprotection stage). It creatively selects and verifies a highly compatible combination of protecting groups and a process route with optimized effects.

[0024] First, to address the instability of ascorbic acid, an NHS active ester (compound I) is prepared in advance. This ester converts the unstable ascorbic acid carboxylic acid into a stable and highly reactive ester, allowing the subsequent coupling reaction with the peptide to be completed at low temperature and in a short time. This greatly reduces the chance of ascorbic acid being exposed to potentially harmful environments during the long coupling reaction.

[0025] Secondly, H-Gly-His(Trt)-Lys(Boc)-OtBu(IIa) was chosen as the coupling partner, with the N-terminus free and matched with the active ester; His was protected by the Trt group, which has large steric hindrance, effectively suppressing the racemization of His during coupling. During removal in TFA, a chemically inert triphenylmethane (Ph3CH) is generated, rather than a destructive carbocation, fundamentally cutting off the source of side reactions. Lys(Boc) & C-terminal -OtBu: These two protecting groups exhibit perfect synchronicity with His(Trt) removal under the same reaction conditions (mild TFA).

[0026] Back-end simultaneous cleaning and removal (achieving high purity): A mild TFA scheme containing a scavenger (such as TIS / water) is employed. This deprotection system can remove Trt, Boc, and OtBu in a single, clean step. The scavenger (such as TIS) can promptly capture any trace cations that may be present, further protecting the product. All molecules after the removal of the protecting group are small volatile molecules or easily removable substances, creating excellent conditions for subsequent purification.

[0027] The interconnected steps of this invention address several key aspects. First, the active ester (I) solves the stability problem of ascorbic acid during the coupling stage (rapid and mild reaction). Second, the specific protecting group (IIa) solves the stability problem during the deprotection stage (avoiding cation attack). Finally, the mild TFA / scavenger system provides a perfect final reaction stage for the two intermediates, achieving simultaneous and clean removal of all protecting groups. It is the combined use of the "active ester method" and the "Trt protecting group strategy" that unlocks the final high yield.

[0028] Beneficial effects

[0029] Compared with the prior art, the present invention has the following significant advantages: [1] High synthesis yield: By pre-preparing a stable succinyl ascorbic acid active ester (compound I) and efficiently coupling it with a protective tripeptide, the degradation of ascorbic acid in the coupling step was reduced, thus increasing the overall yield; [2] High product purity: The optimized protecting group strategy and reaction conditions effectively inhibited racemization and the generation of by-products, simplified the purification process, and the final product purity reached more than 98%.

[0030] [3] Mild reaction conditions: The key coupling reaction was carried out at low temperature to room temperature (-10~25°C), avoiding the destruction of the ascorbic acid structure by high temperature. Deprotection was carried out at room temperature using a mild trifluoroacetic acid / scavenger mixture (such as TFA / TIS / H2O). The entire process avoided harsh conditions such as high temperature, strong acid / strong alkali, etc., and protected the integrity of the enediol active structure and peptide chain of ascorbic acid to the greatest extent. The 1H NMR spectrum of the product showed that the characteristic proton signal of ascorbic acid was clear and there was no oxidative degradation peak.

[0031] [4] Simple operation and easy to scale up: The synthetic route is reasonably designed, the intermediates are stable, and the post-processing of each step is relatively simple, making it suitable for scale-up from laboratory gram-level preparation to industrial kilogram-level production. Attached Figure Description

[0032] Figure 1 The HPLC chromatogram of compound I in Example 1 shows the following peaks: Peak 1 (approximately 3.2 min): trace amounts of ascorbic acid degradation products; Peak 2 (approximately 5.8 min): trace amounts of unreacted intermediate succinyl ascorbic acid; Main peak (approximately 8.5 min): target product NHS-succinyl ascorbate (compound I); Peak 3 (approximately 10.8 min): trace amounts of bissuccinylation byproducts. Figure 2 H-NMR nuclear magnetic resonance of the product in Example 1; Figure 3 C-NMR nuclear magnetic resonance of the product in Example 1; Figure 4 UV-VIS of the product in Example 1; a concentration of c = 5.0 × 10⁻⁶ was prepared. -5 The solution was measured using a standard 1 cm path length cuvette. Figure 5 The IR of the product in Example 1 was obtained by preparing a solution with c = 0.001 mol / L and measuring it using a liquid cell with b = 0.1 cm (thickness of the infrared liquid cell). Figure 6 Raman of the product in Example 1. Detailed Implementation

[0033] Example 1: Preparation of succinyl ascorbic acid tripeptide-1 [Step 1] Preparation of Compound I (NHS-activated succinyl ascorbate) Under nitrogen protection and in the dark, L-ascorbic acid (17.62 g, 100 mmol) was dissolved in 200 mL of anhydrous N,N-dimethylformamide (DMF), and the solution was cooled to 0°C in an ice-salt bath. Triethylamine (30.3 mL, 220 mmol), 4-dimethylaminopyridine (DMAP, 0.61 g, 5 mmol), and succinic anhydride (11.01 g, 110 mmol) were added slowly in sequence with stirring. The ice bath was removed, and the reaction mixture was slowly brought to room temperature (approximately 25°C) and stirred for 4 hours. TLC (developing solvent: dichloromethane / methanol = 8 / 2, v / v) showed that L-ascorbic acid had essentially disappeared. The reaction solution was cooled again to 0°C, and N-hydroxysuccinimide (NHS, 12.65 g, 110 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 21.08 g, 110 mmol) were added sequentially. After stirring at 0°C for 1 hour, the ice bath was removed, and stirring was continued overnight (approximately 12 hours) at room temperature. After the reaction was complete, the reaction solution was poured into 800 mL of ice water, and a solid precipitated. It was extracted with ethyl acetate (3 × 300 mL), and the organic phases were combined and washed sequentially with 5% dilute hydrochloric acid (200 mL), saturated sodium bicarbonate solution (200 mL), and saturated brine (200 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure at 35°C to give a pale yellow crude solid. It was washed with a small amount of cold isopropyl ether and dried under vacuum to give compound I (white to off-white powder).

[0034] The HPLC chromatogram of the product is as follows Figure 1 As shown, peak 1 (approximately 3.2 min): trace amounts of ascorbic acid degradation products; peak 2 (approximately 5.8 min): trace amounts of unreacted intermediate succinyl ascorbic acid; main peak (approximately 8.5 min): target product NHS-succinyl ascorbate (compound I); peak 3 (approximately 10.8 min): trace amounts of bissuccinylation byproducts.

[0035] [Step 2] Preparation of compound IIa (H-Gly-His(Trt)-Lys(Boc)-OtBu) (liquid-phase fragment condensation method) a) Synthesis of Fmoc-Lys(Boc)-OtBu: Fmoc-Lys(Boc)-OH (10.0 g, 20.2 mmol) was dissolved in 100 mL of anhydrous dichloromethane (DCM) and cooled in an ice bath. N,N-diisopropylethylamine (DIPEA, 7.0 mL, 40.4 mmol) was added, followed by the slow dropwise addition of isobutyl chloroformate (2.8 mL, 21.2 mmol). After stirring for 30 minutes, tert-butanol (4.0 mL, 42.4 mmol) and DMAP (catalytic amount) were added. The ice bath was removed, and the reaction mixture was allowed to react overnight at room temperature. The reaction solution was washed with 1M hydrochloric acid, saturated sodium bicarbonate solution, and saturated brine, dried, concentrated, and purified by rapid column chromatography to give a white solid.

[0036] b) De-Fmoc removal: The above product was dissolved in 30 mL of DCM, 10 mL of piperidine was added, and the mixture was stirred at room temperature for 30 minutes. After the reaction was completed by TLC monitoring, the mixture was concentrated under reduced pressure, and recrystallized with ethyl acetate / n-hexane to obtain H-Lys(Boc)-OtBu.

[0037] c) Coupling of the His fragment: H-Lys(Boc)-OtBu (1.0 eq), Fmoc-His(Trt)-OH (1.05 eq), and 1-hydroxybenzotriazole (HOBt, 1.05 eq) were dissolved in anhydrous DMF / DCM mixed solvent and cooled in an ice bath. DIPEA (2.5 eq) was added, followed by the addition of 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HATU, 1.05 eq) in portions. The reaction was carried out at room temperature for 4 hours. After the reaction was complete, the mixture was quenched with water, extracted with ethyl acetate, washed, dried, concentrated, and purified by column chromatography to obtain Fmoc-His(Trt)-Lys(Boc)-OtBu.

[0038] d) Repeat the process of de-Fmoc and coupling with Gly: De-Fmoc is removed as in step b), and then coupled with Boc-Gly-OH (or Fmoc-Gly-OH) as in step c).

[0039] e) Removal of the N-terminal protecting group: If Boc-Gly is used, the Boc is removed using TFA / DCM; if Fmoc-Gly is used, the Fmoc is removed using piperidine. The target compound IIa is finally obtained as a white, foamy solid. The final product is obtained after three steps of coupling and deprotection.

[0040] [Step 3] Synthesis of the target compound (standard conditions) Compound I (4.56 g, 12.5 mmol) prepared in the first step was dissolved in 50 mL of anhydrous dichloromethane (DCM), cooled in an ice bath (0°C), and stirred. Under nitrogen protection, 50 mL of anhydrous DCM solution of compound IIa (H-Gly-His(Trt)-Lys(Boc)-OtBu, 10.0 g, 11.9 mmol) prepared in the second step was added dropwise. After the addition was complete, N,N-diisopropylethylamine (DIPEA, 4.15 mL, 23.8 mmol) was slowly added dropwise. The reaction was stirred at 0°C for 2 hours, and then slowly raised to room temperature (25°C) for another 12 hours. HPLC monitoring (C18 column, acetonitrile / water gradient) showed that the peak area of ​​compound IIa was less than 1%.

[0041] The reaction solution was quenched with 100 mL of saturated ammonium chloride solution, and the mixture was separated. The aqueous phase was back-extracted with DCM (2 × 50 mL). The combined organic phases were washed successively with 1 M citric acid solution, saturated sodium bicarbonate solution, and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure at 35°C to obtain a pale yellow, oily crude protective intermediate.

[0042] The crude product was used directly for the next step of deprotection. It was dissolved in 50 mL of a deprotection mixture (trifluoroacetic acid (TFA) / water / triisopropylsilane (TIS) = 95:2.5:2.5, v / v / v), stirred in an ice bath for 30 minutes, then the ice bath was removed, and the reaction was allowed to proceed at room temperature for 3 hours. HPLC monitoring showed complete removal of the protecting group. The reaction solution was concentrated to near dryness under reduced pressure at 30°C, and 50 mL of cold diethyl ether (0°C) was added with vigorous stirring, resulting in the precipitation of a white solid. The solid was centrifuged, the supernatant was discarded, and the solid was washed three times with cold diethyl ether (3 × 20 mL), and dried under vacuum to obtain a white crude product powder.

[0043] The crude product was purified by preparative high-performance liquid chromatography (preparative HPLC): C18 reversed-phase column, mobile phase A was 0.1% TFA aqueous solution, mobile phase B was acetonitrile, gradient elution (20-50% B, 30 min), detection wavelength 220 nm. The main peak fraction was collected, combined, and freeze-dried to obtain the target compound—succinyl ascorbic acid tripeptide-1—as a white, fluffy powder.

[0044] Example 2: Optimization of coupling solvent (using DMF) The operating procedures are the same as in Example 1, except that the solvent for the coupling reaction is replaced with anhydrous DMF instead of anhydrous DCM, while the amount of DIPEA remains unchanged. The reaction time is shortened to 8 hours. For post-treatment, the reaction solution is concentrated and poured into ice water, then extracted with ethyl acetate; subsequent steps are the same.

[0045] Example 3: Optimization of coupling base (using N-methylmorpholine, NMM) The operating steps are the same as in Example 1, except that DIPEA is replaced with an equimolar amount of N-methylmorpholine (NMM).

[0046] Example 4: Optimized deprotection formulation (using different scavengers) The operating steps are the same as in Example 1, except that in the deprotection step, the deprotection mixture is changed to TFA / water / 1,2-ethylenedithiol (EDT) = 94:3:3 (v / v / v).

[0047] Example 5: Using different combinations of protecting groups (compound IIb: H-Gly-His(Pbf)-Lys(Fmoc)-OtBu) This embodiment aims to illustrate that when using a combination of protection bases different from the preferred scheme, it is necessary to adjust the corresponding deprotection strategy.

[0048] 1. Preparation of compound IIb (H-Gly-His(Pbf)-Lys(Fmoc)-OtBu) The liquid-phase fragment condensation method of Example 1 is used, but with the following substitutions: a) Replace Fmoc-Lys(Boc)-OH with Fmoc-Lys(Fmoc)-OH and react with tert-butanol to generate Fmoc-Lys(Fmoc)-OtBu.

[0049] b) After removing the N-terminal Fmoc, it is coupled with Fmoc-His(Pbf)-OH (replacing Fmoc-His(Trt)-OH) to obtain Fmoc-His(Pbf)-Lys(Fmoc)-OtBu.

[0050] c) After removing the N-terminal Fmoc, it is coupled with Boc-Gly-OH to obtain Boc-Gly-His(Pbf)-Lys(Fmoc)-OtBu.

[0051] d) The N-terminal Boc protecting group was removed by treating the mixture of trifluoroacetic acid / dichloromethane (TFA / DCM, 1:4, v / v) at 0°C for 30 minutes. After concentration under reduced pressure and precipitation with diethyl ether, the target compound IIb (H-Gly-His(Pbf)-Lys(Fmoc)-OtBu) was obtained as a white, foamy solid.

[0052] 2. Coupling reaction with compound I The reaction was carried out under the standard conditions of Example 1: Compound I (12.5 mmol) and Compound IIb (11.9 mmol) were coupled in anhydrous DCM at 0°C in the presence of DIPEA (23.8 mmol). The reaction progress was monitored by HPLC. The post-processing method was the same as in Example 1, yielding a crude product of the protected intermediate Boc-Gly-His(Pbf)-Lys(Fmoc)-OtBu (assuming that Pbf and Fmoc are retained after coupling).

[0053] 3. Step-by-step deprotection Since the protecting group combination includes base-sensitive Fmoc and acid-sensitive Pbf and OtBu, a stepwise deprotection strategy is required: a) Removal of the Lys side-chain Fmoc protecting group: The crude product obtained in the previous step was dissolved in N,N-dimethylformamide (DMF), and a 20% (v / v) piperidine DMF solution was added. The mixture was stirred at room temperature for 30 minutes. The removal of Fmoc was monitored by TLC or HPLC until complete removal. The reaction solution was concentrated under reduced pressure, and the residue was dissolved in ethyl acetate and washed successively with 0.5 M hydrochloric acid, saturated sodium bicarbonate solution, and saturated brine. The organic phase was dried over anhydrous sodium sulfate and then concentrated to obtain the intermediate with a free side chain.

[0054] b) Acid deprotection to remove residual protecting groups (Pbf, OtBu): The above intermediate was dissolved in a deprotection mixture (trifluoroacetic acid (TFA) / water / triisopropylsilane (TIS) = 95:2.5:2.5, v / v / v) and stirred at room temperature for 3-4 hours. HPLC monitoring showed complete removal of the protecting groups.

[0055] c) Post-treatment and purification: The reaction solution was concentrated to near dryness under reduced pressure, and cold diethyl ether was added to precipitate the solid. Subsequent precipitation, washing, drying, and preparative HPLC purification steps were exactly the same as in Example 1.

[0056] Example 6: Hundred-gram scale-up experiment Following the optimal conditions of Example 1 (DCM as solvent, DIPEA as base, TFA / TIS / water deprotection), the feed amounts were scaled up proportionally: compound IIa (100.0 g, 119 mmol), compound I (46.0 g, 125 mmol), DIPEA (41.5 mL), and DCM (1.0 L). The reaction was carried out in a 20 L glass reactor with well-controlled temperature and stirring. For post-processing, vacuum distillation was used instead of rotary evaporation for concentration. Purification was performed in batches using a medium-pressure preparative chromatography system instead of analytical preparative HPLC.

[0057] Comparative Example 1: Traditional one-pot coupling (without pre-preparation of the active ester)

[0058] Succinyl ascorbic acid (intermediate, prepared according to Example 1 but not as an NHS ester), compound IIa (11.9 mmol), HOBt (1.61 g, 11.9 mmol), and EDC·HCl (2.28 g, 11.9 mmol) were dissolved in anhydrous DMF (100 mL) and cooled in an ice bath. DIPEA (4.15 mL) was added. The mixture was stirred at room temperature for 24 hours. HPLC monitoring showed that a large amount of compound IIa remained (>40%). Forced post-processing and deprotection were performed, followed by purification.

[0059] Comparative Example 2: Deprotection using strong acid / strong conditions After obtaining the protected intermediate according to Example 1, instead of using the mild TFA / TIS / water system, deprotection was performed using a strongly acidic "cutting agent A" (a mixture of hydrofluoric acid / anisole / p-cresol, for cutting conditions of strongly acid-sensitive resins). The reaction was carried out at 0°C for 1 hour.

[0060] Comparative Example 3: High-Temperature Coupling Reaction The operating steps are the same as in Example 1, but the coupling reaction is carried out in a 40°C oil bath.

[0061] Comparative Example 4: Using different active esters (p-nitrophenol ester) Following a method similar to Example 1, but replacing NHS with p-nitrophenol, succinyl ascorbate p-nitrophenol ester was prepared. It was then coupled with compound IIa under the conditions of Example 1.

[0062] Comparative Example 5: Inappropriate selection of protecting group (His side chain protected with Boc) An attempt was made to synthesize compound IIc: H-Gly-His(Boc)-Lys(Boc)-OtBu. The coupling reaction proceeded normally (under the same conditions as in Example 1). However, during the subsequent acid deprotection step (using TFA / TIS / water), HPLC monitoring revealed numerous complex byproducts.

[0063] Comparative Example 6: Coupling under alkali-free conditions The operation steps are the same as in Example 1, but the addition of DIPEA is omitted.

[0064] Test Results Synthesis Yield Test Test methods: HPLC chromatograph, high performance liquid chromatography (HPLC).

[0065] Instrument: High-performance liquid chromatography system equipped with a diode array detector (DAD).

[0066] Chromatographic column: Agilent ZORBAX SB-C18 column, 4.6 × 250 mm, particle size 5 μm, or equivalent column.

[0067] Mobile phase: Phase A: 0.1% (v / v) trifluoroacetic acid (TFA) aqueous solution.

[0068] Phase B: 0.1% (v / v) trifluoroacetic acid (TFA) acetonitrile solution.

[0069] Elution procedure: Gradient elution is used, as shown in Table 1.

[0070] Table 1:

[0071] Column temperature: 30 °C Detection wavelength: 220 nm (for detecting peptide bonds and ascorbic acid backbone).

[0072] Injection volume: 10 μL.

[0073] Sample preparation: Accurately weigh approximately 2.0 mg of the purified lyophilized sample and place it in a 10 mL volumetric flask. Dissolve the sample in a mixed solvent with an initial mobile phase ratio (A phase / B phase = 95 / 5, v / v) and bring the volume to the mark. After sonication to aid dissolution, filter the solution through a 0.22 μm organic filter membrane and use the filtrate as the test solution. The results are shown in Table 2.

[0074] Table 2

[0075] The synthesis method of this invention exhibits significant technical superiority and reproducibility. The final yields of all embodiments remained consistently high, ranging from 48% to 78%, with the optimized Example 1 reaching 78%. Furthermore, the yield in the scale-up experiment (Example 6) remained at 50%, demonstrating the excellent scalability and stability of this process route. In contrast, the conventional or suboptimal methods represented by the comparative examples generally had low yields. Comparative Example 2 (strong acid deprotection) and Comparative Example 6 (base-free coupling) had yields of only 15% and 5%, respectively, while Comparative Example 1 (conventional one-pot method) had a yield of only 22%, all significantly lower than the levels of the embodiments of this invention. Crucially, Comparative Example 5 (using an inappropriate protecting group) had a yield below 10%, directly confirming the necessity of the specific protecting group strategy in this invention. In summary, this invention, through a synergistic technical solution of pre-preparing stable active esters, optimizing the protecting group combination, and employing mild reaction conditions, systematically solves the core defects of existing technologies, such as low yields, numerous side reactions, and harsh conditions, more than doubling the synthesis efficiency and achieving unexpected technical effects.

Claims

1. A method for synthesizing succinyl ascorbic acid tripeptide-1, characterized in that, Includes the following steps: (1) L-ascorbic acid is reacted with succinic anhydride to generate succinyl ascorbic acid, which is then reacted with N-hydroxysuccinimide in the presence of a condensing agent to obtain succinyl ascorbic acid active ester (compound I). (2) Prepare a tripeptide-1 derivative (compound II) with a side chain and C-terminus protected by a protecting group and an N-terminus of a free amino group, wherein the sequence of the tripeptide-1 is Gly-His-Lys; (3) In the presence of an organic base, compound I obtained in step (1) and compound II obtained in step (2) are coupled in an aprotic solvent to obtain a protected succinyl ascorbic acid tripeptide-1 intermediate. (4) Remove all protecting groups from the intermediate obtained in step (3) and purify to obtain succinyl ascorbic acid tripeptide-1.

2. The method according to claim 1, characterized in that, In step (1), the condensing agent is one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) or dicyclohexylcarbodiimide (DCC); the reaction is carried out in an aprotic solvent at a temperature of 0-25°C.

3. The method according to claim 1, characterized in that, In step (2), the protecting group is independently selected from: the amino protecting group is fluorenyl methoxycarbonyl (Fmoc) or tert-butyl methoxycarbonyl (Boc); the protecting group of the imidazole nitrogen of the histidine side chain is triphenylmethyl (Trt) or 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf); the protecting group of the ε-amino of the lysine side chain is tert-butyl methoxycarbonyl (Boc) or fluorenyl methoxycarbonyl (Fmoc); and the carboxyl protecting group is tert-butyl ester (OtBu) or benzyl ester (OBn).

4. The method according to claim 1, characterized in that, In step (2), the preferred structure of compound II is H-Gly-His(Trt)-Lys(Boc)-OtBu or H-Gly-His(Pbf)-Lys(Fmoc)-OtBu.

5. The method according to claim 1, characterized in that, In step (3), the organic base is N,N-diisopropylethylamine (DIPEA) or N-methylmorpholine (NMM); the aprotic solvent is one or more of dichloromethane (DCM), N,N-dimethylformamide (DMF), and tetrahydrofuran (THF).

6. The method according to claim 1, characterized in that, In step (3), the coupling reaction temperature is from -10°C to 25°C.

7. The method according to claim 1, characterized in that, In step (4), the deprotection is performed using a mixture of trifluoroacetic acid (TFA) and a scavenging agent, wherein the scavenging agent is one or more of water, triisopropylsilane (TIS), anisole, and 1,2-ethylenedithiol.

8. The method according to claim 6, characterized in that, The volume fraction of TFA in the mixture is 90-97%, and the total volume fraction of the cleaning agent is 3-10%.

9. The method according to claim 1, characterized in that, In step (4), the purification is carried out by reversed-phase high-performance liquid chromatography, using acetonitrile-water solution containing 0.1% trifluoroacetic acid as the mobile phase for gradient elution.

10. Succinyl ascorbic acid tripeptide-1 prepared by the method according to any one of claims 1 to 9.

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