Biomimetic catalyst based on MOF material, preparation method of biomimetic catalyst and application of biomimetic catalyst in peptide synthesis

Abstract

The invention discloses a biomimetic catalyst based on an MOF material, a preparation method of the biomimetic catalyst and application of the biomimetic catalyst in peptide synthesis. According to the method, firstly, a novel functional ligand is designed and synthesized, and the ligand integrates nucleophilic sulfydryl, proton transfer imidazolyl and skeleton connection carboxyl and can simulate a'catalytic triad 'of an enzyme active center. Then, the ligand, metal salt and terephthalic acid are co-assembled through a solvothermal method, and the bionic MOF material is prepared. The material has regular nanopore channels and high stability, and can accurately simulate the catalytic mechanism of cysteine protease. The catalyst is used for catalyzing peptide synthesis reaction, amino-acid ester condensation can be efficiently catalyzed to form peptide bonds under the mild and green conditions without chemical coupling agents, the conversion rate is high, the catalyst is easy to recycle, the inherent defects of a traditional chemical synthesis method and an enzyme method are overcome, and a new strategy is provided for green peptide synthesis.

Description

Technical Field

[0001] This invention pertains to the organic synthesis of biomimetic catalytic materials, specifically relating to a biomimetic catalyst based on MOF materials, its preparation method, and its application in peptide synthesis. Background Technology

[0002] Peptides are key functional molecules involved in life processes, composed of two or more amino acids linked by peptide bonds, and play a vital role in pharmaceutical development, biotechnology, functional foods, and cosmetics. With the booming development of synthetic biology and the peptide drug market, the demand for efficient, green, and low-cost peptide synthesis technologies is increasingly urgent. Currently, commercial peptide synthesis mainly relies on chemical methods, particularly solid-phase peptide synthesis (SPPS). However, this method has inherent and serious drawbacks. It requires excessive use of expensive and often toxic chemical coupling agents (such as carbodiimide reagents) and activating agents, resulting in extremely low atom economy and generating large amounts of difficult-to-treat chemical waste after each coupling step, posing a serious threat to the environment. Furthermore, lengthy purification steps and the potential risk of amino acid racemization significantly increase production costs and technical complexity, which contradicts the concept of sustainable green chemistry. To overcome the drawbacks of chemical synthesis, enzymatic synthesis routes utilizing proteases as biocatalysts offer a highly attractive alternative. Enzymatic synthesis offers mild reaction conditions, high specificity and selectivity, and is environmentally friendly. Cysteine ​​proteases (such as papain) exhibit great potential in peptide synthesis due to their unique catalytic mechanism. The core of their catalytic activity lies in a "catalytic triplet" precisely arranged from cysteine ​​(Cys), histidine (His), and aspartic acid (Asp) residues. In this structure, the thiol group of the Cys residue acts as a strong nucleophile, responsible for attacking the carbonyl carbon of the substrate; the imidazole ring of the His residue acts as a proton shuttle medium, serving as a generalized acid-base catalyst to assist in proton transfer; and the carboxyl group of the Asp residue finely regulates the pKa value of His through a hydrogen bond network and stabilizes the transition state. The synergistic effect of these three components in space forms the basis for highly efficient catalysis of peptide bond formation.

[0003] Despite the significant advantages of enzymatic synthesis, the inherent instability of natural proteases is a major obstacle to their industrial application. Natural enzymes are extremely sensitive to operating conditions, readily denaturing and losing activity in high temperatures, extreme pH levels, or organic solvent environments. Furthermore, their production and purification processes are complex and costly, and their separation and recovery from the reaction system are difficult, hindering reusability. These factors collectively limit their widespread application in large-scale peptide synthesis. Summary of the Invention

[0004] Objective of this invention: The objective of this invention is to provide a biomimetic catalyst based on MOF materials that mimics the catalytic activity of cysteine ​​proteases. This biomimetic catalyst precisely mimics the "catalytic triplet" structure and synergistic catalytic mechanism of the enzyme's active site by covalently introducing functional ligands integrating thiol, imidazole, and carboxyl groups into a MOF framework with specific pore sizes. Another objective of this invention is to provide a method for preparing this biomimetic catalyst and its application in catalyzing peptide synthesis reactions efficiently and selectively under mild, coupling agent-free conditions.

[0005] Technical solution: The biomimetic catalyst based on MOF materials described in this invention comprises zinc or zirconium metal nodes and functional ligands containing thiol, imidazole, and carboxyl groups, with an opening size of [missing information]. Cys-His-Asp triplet for mimicking the active site of cysteine ​​proteases; the functional ligand is 2-((1H-imidazol-4-ylmethyl)amino)-5-mercaptoterephthalic acid or a derivative thereof.

[0006] The preparation method of the biomimetic catalyst based on MOF material of the present invention includes preparing a functional ligand, dissolving a metal salt, terephthalic acid and the functional ligand in a solvent in a certain proportion, adding a structure modifier, dispersing by ultrasound, and then carrying out a solvothermal reaction; after the reaction is completed, the product is collected by centrifugation, washed with organic solvent, solvent exchanged and activated by high temperature vacuum to obtain the biomimetic catalyst based on MOF material.

[0007] Preferably, the preparation method of the functional ligand is as follows: 2,5-dinitroterephthalic acid is reacted with methanol under reflux catalysis of concentrated sulfuric acid to generate dimethyl 2,5-dinitroterephthalate; the dinitro ester is hydrogenated and reduced to generate dimethyl 2,5-diaminoterephthalate; the diamino ester is reacted with 4-(chloromethyl)-1H-imidazolium hydrochloride to introduce an imidazolium group, yielding a monosubstituted product; the monosubstituted product is subjected to a diazotization reaction, and then reacted with sodium hydrosulfide to introduce a thiol group; the thiol-containing ester is hydrolyzed to generate the target product IMTA ligand (2-((1H-imidazol-4-ylmethyl)amino)-5-mercaptoterephthalic acid).

[0008] A further preferred method for preparing the functional ligand is as follows: First, 2,5-dinitroterephthalic acid is added to excess anhydrous methanol, and concentrated sulfuric acid is used as a catalyst (5-15% of the molar amount of the raw material). The mixture is heated under reflux at 60-80°C for 12 to 24 hours. After the reaction is complete, a white solid precipitates upon cooling. This solid is filtered, washed, and dried to obtain dimethyl 2,5-dinitroterephthalate. Subsequently, the product from the previous step is dissolved in methanol or ethyl acetate, and 5-10% Pd / C (5% or 10% loading) is added as a catalyst. The mixture is stirred and reacted at a hydrogen pressure of 0.1 to 0.5 MPa and a temperature of 25 to 50°C for 12 to 36 hours until no more hydrogen is consumed. After the reaction is complete, the Pd / C catalyst is removed by diatomaceous earth filtration, and the solvent is removed by rotary evaporation to obtain dimethyl 2,5-diaminoterephthalate. Next, the diamino product obtained in the previous step is dissolved in N,N-dimethylformamide (DMF) or acetonitrile, and 1.0 to 1.2 equivalents of 4-(chloromethyl)-1H-imidazolium hydrochloride and 2.0 to 3.0 equivalents of an inorganic base (such as potassium carbonate) or an organic base (such as triethylamine) are added. The mixture is stirred at 40 to 70 °C for 24 to 48 hours. The reaction solution is purified by extraction, drying, and column chromatography (eluent system: dichloromethane / methanol) to obtain the monosubstituted product 2-amino-5-((1H-imidazol-4-ylmethyl)amino)dimethyl terephthalate. Then, the product from the previous step is dissolved in a 1-5 M aqueous solution of hydrochloric acid or sulfuric acid, cooled to 0-5 °C, and 1.0 to 1.2 equivalents of an aqueous solution of sodium nitrite is slowly added dropwise under vigorous stirring. The reaction is maintained at this temperature for 1 to 2 hours to generate the diazonium salt. Subsequently, this diazonium salt solution is slowly added dropwise to a pre-prepared, excess aqueous solution of sodium hydrosulfide or potassium ethyl xanthate, and the reaction is carried out at 5 to 25°C for 2 to 5 hours. After the reaction is complete, a thiol-containing esterified product is obtained. Finally, the obtained crude product containing thiol is dissolved in a methanol / water mixture, and 3 to 10 equivalents of sodium hydroxide or potassium hydroxide are added. The mixture is heated to 60-90°C and refluxed for 4 to 12 hours to completely hydrolyze the methyl ester. After the reaction is complete, the mixture is cooled to room temperature, and the pH is carefully adjusted to 2.5-4.0 with 2-6M hydrochloric acid. At this point, the target product IMTA will precipitate out as a solid. The precipitate was collected by vacuum filtration and repeatedly washed with deionized water and a small amount of ethanol to remove inorganic salts. Finally, it was vacuum dried at 60-80℃ for more than 12 hours to obtain a white solid biomimetic multifunctional ligand integrating a nucleophilic thiol group, a proton-transfer imidazole group, and a carboxyl group linked to the skeleton, namely 2-((1H-imidazol-4-ylmethyl)amino)-5-mercaptoterephthalic acid (IMTA), or IMTA ligand.

[0009] Preferably, the metal salt is zirconium tetrachloride (ZrCl4) or zinc nitrate (Zn(NO3)2).

[0010] Preferably, the molar ratio of the metal salt, terephthalic acid, and functional ligand is 1:(0.8-0.98):(0.02-0.2).

[0011] Preferably, the structure modifier is acetic acid or formic acid, and its addition amount is 1% to 10% of the total solvent volume.

[0012] Preferably, the temperature of the solvothermal reaction is 100-140°C and the time is 24-72 hours.

[0013] In a further preferred embodiment, the metal salt, terephthalic acid, and functional ligand are added at a molar ratio of 1:(0.8-0.98):(0.02-0.2) and dispersed in N,N-dimethylformamide, with the total ligand concentration controlled at 0.01-0.1M. Acetic acid or formic acid (1%-10% by volume) is added as a structure modifier, and after ultrasonic dispersion, the mixture is transferred to a Teflon-lined reactor and crystallized at 100-140℃ for 24-72 hours. After natural cooling, the initial product is collected by centrifugation, washed 3-5 times with fresh DMF at 80-100℃, and exchanged with anhydrous ethanol or acetone at least 3 times. Finally, it is activated under vacuum at 120-150℃ for 12-24 hours to obtain a biomimetic catalyst based on MOF material.

[0014] Further preferably, the metal salt, terephthalic acid, and functional ligand IMTA are fed in a strict molar ratio of 1:(0.8-0.98):(0.02-0.2). All solid raw materials are dispersed in N,N-dimethylformamide (DMF) solvent, so that the total concentration of the ligand is controlled between 0.01M and 0.1M. To regulate crystal nucleation and growth, acetic acid or formic acid as a structure regulator is added to the mixed solvent, accounting for 1% to 10% of the total solvent volume (or 10 to 100 times the molar amount of the metal salt). After the mixture is ultrasonically dispersed evenly, it is transferred to a Teflon-lined stainless steel reactor, sealed, and placed in an oven for crystallization at a constant temperature of 100 to 140°C for 24 to 72 hours. After the reaction is completed, the reactor is allowed to cool naturally to room temperature, and the initial product is collected by high-speed centrifugation (8000-12000 rpm). To thoroughly remove unreacted reactant and DMF guest molecules from the pores, the product was repeatedly washed with fresh DMF at 80-100°C 3 to 5 times, each time for no less than 12 hours. Subsequently, solvent exchange was performed at least 3 times with anhydrous ethanol or acetone to displace the high-boiling-point DMF. Finally, the sample was activated at 120-150°C and under high vacuum (pressure below 10 Pa) for 12 to 24 hours to obtain a biomimetic catalyst based on MOF materials.

[0015] The application of the MOF-based biomimetic catalyst in peptide synthesis described in this invention is as follows: under the condition of no chemical coupling agent, the MOF-based biomimetic catalyst is used to catalyze the condensation reaction of amino acid esters to generate peptide bonds.

[0016] Preferably, the catalytic reaction temperature is 30-60°C, and the reaction time is 12-48 hours; the amount of catalyst used is 5% to 20% of the initial mass of the amino acid ester.

[0017] A further preferred application method is as follows: In a dry reaction flask under nitrogen or argon protection, using amino acid esters (methyl, ethyl, or benzyl esters) as the sole substrate, a biomimetic catalyst based on MOF materials is added. A strictly dry, low-water-content organic solvent (acetonitrile, tetrahydrofuran, or dichloromethane) is used, controlling the substrate concentration at 0.05-0.2 M. The mixture is magnetically stirred at 30-60°C for 12-48 hours (no additional chemical coupling agent or pH adjustment is required). After the reaction, the solid catalyst is separated by centrifugation or filtration. The catalyst can be recycled after washing with ethanol / ethyl acetate and vacuum drying at 60-80°C. The filtrate is distilled under reduced pressure to remove the solvent, yielding the crude product.

[0018] A further preferred application method is as follows: In a dry reaction flask filled with nitrogen or argon, an amino acid ester (such as methyl ester, ethyl ester, or benzyl ester) is added as the sole substrate. A biomimetic catalyst based on MOF materials is then added to the reaction system. A strictly dried, low-water-content organic solvent (such as acetonitrile, tetrahydrofuran, or dichloromethane) is used, with the substrate concentration controlled between 0.05 M and 0.2 M. The reaction mixture is magnetically stirred at a mild temperature of 30 to 60 °C for 12 to 48 hours. No additional chemical coupling agents or pH adjustments are required throughout the catalytic process. After the reaction, the solid catalyst can be separated and recovered from the reaction solution by simple centrifugation (5000-10000 rpm) or filtration. The recovered catalyst is washed several times with ethanol or ethyl acetate and then vacuum dried at 60-80 °C before being directly used in the next reaction cycle. The combined filtrates were distilled off under reduced pressure to remove the solvent. The crude product was purified by silica gel column chromatography (e.g., using a gradient-eluting hexane / ethyl acetate or dichloromethane / methanol mixture) or recrystallization to finally obtain the target dipeptide ester product with high purity (>95%).

[0019] Preferably, after the reaction is completed, the catalyst is recovered by centrifugation or filtration. The recovered catalyst can be reused for the next catalytic reaction after washing and drying. The reaction solution is concentrated and purified by column chromatography or recrystallization to obtain the target peptide product.

[0020] A further preferred method for silica gel column chromatography purification is as follows: Use 200-300 mesh or 300-400 mesh silica gel specifically designed for column chromatography. Dissolve the crude product in a minimal amount of highly polar solvent (such as dichloromethane or ethyl acetate), or mix thoroughly with a small amount of silica gel (approximately 1-2 times the mass of the crude product), evaporate to dryness, and then evenly spread the solution at the top of the silica gel column. For non-polar or moderately polar products, a hexane / ethyl acetate system can be used. The initial eluent is low-polarity hexane (containing 1-5% ethyl acetate), used to wash away non-polar impurities. Subsequently, gradually increase the proportion of ethyl acetate, for example, starting with a volume ratio of 98:2, gradually transitioning to 95:5, 90:10, 80:20, until the product is completely eluted. The elution flow rate is controlled at 1-5 mL / min. For more polar products, a dichloromethane / methanol system can be used. The initial eluent is pure dichloromethane or dichloromethane containing 0.5-1% methanol. The proportion of methanol is then gradually increased, for example, from a volume ratio of 100:1 to 50:1, 20:1, until the target product is eluted. The collected fractions are monitored in real-time by thin-layer chromatography (TLC), and fractions containing the target product are combined. The combined purified fractions are then evaporated at 30-40°C using a rotary evaporator to remove the solvent, yielding the target peptide ester product with a purity greater than 95%, in a yield of 35-85%.

[0021] A further preferred method for recrystallization purification involves the following steps: The crude product is placed in an Erlenmeyer flask, and a calculated amount of a good solvent (dichloromethane, ethanol, etc.) is added. The mixture is heated (50-80°C) and stirred until completely dissolved, forming a saturated or nearly saturated solution. If the solution is colored or not clear, a small amount of activated carbon (approximately 1-2% of the crude product mass) can be added for decolorization, and the color is removed by hot filtration. The hot, clear filtrate is allowed to stand at room temperature to cool slowly. To promote crystal growth, the solution can be further refrigerated at 0-4°C for 2 to 12 hours. After complete crystal precipitation, the crystals are collected by vacuum filtration, and the crystal surface is washed with a small amount of pre-cooled recrystallization solvent (-10 to 0°C) to remove residual mother liquor. Finally, the collected crystals are dried in a vacuum oven at 40-60°C for 6 to 12 hours to obtain a product with a purity greater than 95%, yielding 55-80%.

[0022] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0023] 1. Biomimetic structure and function with a clear catalytic mechanism: This invention successfully constructs an active center that mimics the "catalytic triplet" of cysteine ​​protease at the molecular scale. Its thiol, imidazole and carboxyl groups can work synergistically within the nanopores, achieving highly efficient biomimetic catalysis.

[0024] 2. High stability and reusability: MOF materials exhibit excellent thermal and chemical stability, far exceeding that of natural proteases. The catalyst is easily separated and recovered from the reaction system.

[0025] 3. Good versatility and broad application prospects: This method can be applied to peptide bond coupling reactions of various amino acid substrates, providing a new strategy for developing a new generation of high-performance and environmentally friendly industrial catalysts. Detailed Implementation

[0026] The technical solution of the present invention will be further described below with reference to the embodiments.

[0027] Example 1

[0028] (1) Preparation of IMTA ligands

[0029] In a 500 mL round-bottom flask, 51.2 g (0.2 mol) of 2,5-dinitroterephthalic acid and 300 mL of anhydrous methanol were added. Concentrated sulfuric acid (15 mL) was slowly added dropwise, and the mixture was then heated to 70 °C and refluxed for 18 hours. After the reaction was complete, the solution was cooled to room temperature, and a large amount of white solid precipitated. The solid was filtered through a Buchner funnel, washed with a small amount of ice-cold methanol, and then dried in a vacuum oven at 60 °C for 12 hours to give dimethyl 2,5-dinitroterephthalate (54.8 g, 97% yield).

[0030] The product obtained in the previous step (54.8 g) and 5.5 g of Pd / C were added to a 500 mL high-pressure reactor, along with 300 mL of methanol. The air in the reactor was purged three times with nitrogen, and then hydrogen was introduced to a pressure of 0.3 MPa. The reaction was vigorously stirred at 40 °C for 24 hours until the pressure no longer decreased. After the reaction was complete, the mixture was cooled to room temperature, and Pd / C was removed by filtration through filter paper covered with diatomaceous earth. The filtrate was then subjected to rotary evaporation under reduced pressure at 40 °C to remove methanol, yielding a pale yellow solid, dimethyl 2,5-diaminoterephthalate (42.5 g, 98% yield).

[0031] Dimethyl 2,5-diaminoterephthalate (42.5 g, 0.19 mol), 4-(chloromethyl)-1H-imidazolium hydrochloride (29.4 g, 0.19 mol), and anhydrous potassium carbonate (78.8 g, 0.57 mol) were added to a 500 mL three-necked flask, along with 300 mL of anhydrous DMF. Under nitrogen protection, the mixture was heated to 60 °C and stirred for 36 hours. After the reaction was complete, the reaction solution was poured into 1 L of water and extracted three times with ethyl acetate (300 mL each time). The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After filtration, the solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by silica gel column chromatography using dichloromethane / methanol (volume ratio gradually increased from 100:1 to 50:1) as eluent to obtain the pure monosubstituted product 2-amino-5-((1H-imidazol-4-ylmethyl)amino)dimethyl terephthalate (35.2 g, yield 61%).

[0032] The product from the previous step (35.2 g) was dissolved in 200 mL of 3M hydrochloric acid aqueous solution, and the mixture was cooled to 0°C. Under vigorous stirring, a saturated aqueous solution of sodium nitrite (8.7 g, 0.126 mol) (approximately 20 mL) was slowly added dropwise, maintaining the temperature between 0 and 5°C. After the addition was complete, the reaction continued for 1.5 hours. In another beaker, sodium hydrosulfide hydrate (NaSH·xH₂O, 25.8 g, approximately 0.46 mol) was dissolved in 150 mL of water and cooled to 10°C. The above diazonium salt solution was slowly added dropwise to the sodium hydrosulfide solution, during which a large amount of nitrogen gas was released. After the addition was complete, the mixture was stirred at 15°C for 3 hours. The solution was neutralized to approximately pH 7 with 6M hydrochloric acid, then extracted with ethyl acetate, dried, and rotary evaporated to obtain a crude ester containing thiol groups. The crude ester was dissolved in a methanol / water mixture (2:1, 300 mL, v / v), and sodium hydroxide (27.6 g, 0.69 mol) was added. The mixture was heated to 80 °C and refluxed for 8 hours. After the reaction was complete, the mixture was cooled to room temperature. Under ice bath conditions, the pH was carefully adjusted to 3.5 with 6 M hydrochloric acid, resulting in the precipitation of a large amount of white precipitate. The precipitate was collected by suction filtration, washed three times with 500 mL of deionized water, and then washed once with 50 mL of ethanol. The obtained solid was dried under vacuum at 70 °C for 24 hours to obtain the final product IMTA (24.1 g, total yield of 74% for both steps).

[0033] (2) Synthesis of biomimetic catalysts based on MOF materials

[0034] In a 100 mL Teflon-lined stainless steel reactor, zirconium tetrachloride (ZrCl4, 233 mg, 1.0 mmol), terephthalic acid (BDC, 149 mg, 0.9 mmol), and the functional ligand IMTA (28.4 mg, 0.1 mmol) were added sequentially. Then, N,N-dimethylformamide (DMF, 50 mL) and acetic acid (5 mL) were added. The mixture was sonicated for 15 minutes to ensure uniform dispersion. The reactor was sealed and heated in an oven at 120 °C for 48 hours.

[0035] After the reaction was complete, the reactor was allowed to cool naturally to room temperature. The product was collected by centrifugation at 10,000 rpm for 5 minutes. The supernatant was discarded, and fresh DMF (50 mL) was added. The mixture was soaked at 90 °C for 12 hours, and this process was repeated 4 times. Then, solvent exchange was performed with anhydrous ethanol (50 mL), soaking for 8 hours each time, for a total of 3 times. Finally, the sample was activated at 130 °C and under high vacuum (<10 Pa) for 18 hours to obtain approximately 350 mg of white powdery biomimetic catalyst based on MOF material.

[0036] (3) The condensation reaction of L-alanine methyl ester catalyzed by a biomimetic catalyst based on MOF material

[0037] In a nitrogen-filled 50 mL Schlenk flask, L-alanine methyl ester hydrochloride (139.5 mg, 1.0 mmol) and triethylamine (0.14 mL, 1.0 mmol) were added to release the free amino acid ester. Strictly dried acetonitrile (10 mL) was added as a solvent, along with a MOF-based biomimetic catalyst (20 mg, 14% of the substrate mass). The reaction mixture was magnetically stirred in an oil bath at 45 °C for 24 hours. After the reaction was complete, the solid catalyst was recovered by centrifugation (8000 rpm, 5 min). The supernatant was transferred to a round-bottom flask, and the solvent was removed by rotary evaporation under reduced pressure at 35 °C to obtain a white solid crude product. The crude product was purified by silica gel column chromatography: 5 g of 300-400 mesh silica gel was weighed and mixed with hexane / ethyl acetate (10:1 v / v) to form a homogenate, which was then wet-packed into a 1.5 cm inner diameter chromatography column. The crude product was dissolved in 1 mL of dichloromethane, mixed with 100 mg of silica gel, evaporated to dryness, and then evenly spread on the top of a column. Elution was first performed with hexane / ethyl acetate (10:1, 100 mL, v / v), then the ethyl acetate ratio was gradually increased to 2:1. The eluent was monitored using a TLC plate (hexane / ethyl acetate 3:1 as the developing solvent). The fractions containing the target product (L-alanyl-L-alanine methyl ester) were combined, and the solvent was removed by rotary evaporation under reduced pressure to give a pure white solid product (78 mg, 83% yield).

[0038] Example 2

[0039] (1) Preparation of IMTA ligands

[0040] In a 1 L three-necked flask, 102.4 g (0.4 mol) of 2,5-dinitroterephthalic acid and 600 mL of methanol were added. Concentrated sulfuric acid (30 mL) was slowly added dropwise while cooling in an ice bath, and the mixture was then refluxed at 80 °C for 24 hours. After the reaction was complete, the solution was cooled to room temperature, and a white solid precipitated. The solid was filtered, washed with ice-cold methanol, and then dried under vacuum at 70 °C to give dimethyl 2,5-dinitroterephthalate (108.5 g, 96% yield).

[0041] The product from the previous step (108.5 g) and 10.8 g of Pd / C were added to a 1 L high-pressure reactor, along with 600 mL of methanol. The reactor was purged with nitrogen three times, and then purged with hydrogen to a pressure of 0.5 MPa. The reaction was vigorously stirred at 50 °C for 36 hours. After the reaction was complete, the reactor was cooled and filtered to remove the catalyst. The filtrate was evaporated under reduced pressure to remove methanol, yielding a pale yellow solid, dimethyl 2,5-diaminoterephthalate (83.2 g, 97% yield).

[0042] Dimethyl 2,5-diaminoterephthalate (83.2 g, 0.37 mol), 4-(chloromethyl)-1H-imidazolium hydrochloride (58.8 g, 0.37 mol), and anhydrous potassium carbonate (154.2 g, 1.12 mol) were added to a 1 L three-necked flask, along with 600 mL of anhydrous DMF. Under nitrogen protection, the mixture was heated to 80 °C and stirred for 48 hours. After the reaction was complete, the reaction mixture was poured into 2 L of water and extracted with ethyl acetate (3 × 500 mL). The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After filtration, the solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol, v / v ratio from 80:1 to 40:1) to give a pure monosubstituted product (65.7 g, yield 58%).

[0043] The product from the previous step (65.7 g) was dissolved in 3M hydrochloric acid (400 mL) and cooled to 0°C. An aqueous solution of sodium nitrite (16.3 g, 0.236 mol) was slowly added dropwise, maintaining the temperature below 5°C, and the reaction was allowed to proceed for 2 hours. This diazonium salt solution was then slowly added dropwise to an aqueous solution of sodium hydrosulfide hydrate (NaSH·xH₂O, 48.2 g, approximately 0.86 mol) at 10°C (300 mL). After the addition was complete, the mixture was stirred at room temperature for 4 hours. The solution was neutralized with 6M hydrochloric acid to a pH of approximately 7, then extracted with ethyl acetate, dried, and rotary evaporated to obtain a crude ester containing thiol groups.

[0044] The crude ester was dissolved in methanol / water (1:1, 600 mL, v / v), and sodium hydroxide (51.6 g, 1.29 mol) was added. The mixture was refluxed at 90 °C for 12 hours. After the reaction was complete, the mixture was cooled to room temperature. Under ice bath conditions, the pH was adjusted to 3.0 with 6 M hydrochloric acid, resulting in the precipitation of a white precipitate. The precipitate was collected by suction filtration, washed three times with 1 L of deionized water, and then washed once with 100 mL of ethanol. The obtained solid was dried under vacuum at 80 °C for 24 hours to obtain the final product IMTA (44.8 g, total yield of 71% for both steps).

[0045] (2) Synthesis of biomimetic catalysts based on MOF materials

[0046] In a 250 mL Lflon-lined reactor, zirconium tetrachloride (ZrCl4, 466 mg, 2.0 mmol), terephthalic acid (BDC, 249 mg, 1.5 mmol), and IMTA ligand (142 mg, 0.5 mmol) were added. N,N-dimethylformamide (DMF, 100 mL) and formic acid (10 mL) were then added. The mixture was sonicated for 20 minutes. The reactor was then sealed and heated in an oven at 130 °C for 72 hours.

[0047] After the reaction was completed, the sample was allowed to cool naturally. The product was collected by centrifugation and soaked in fresh DMF (100 mL) at 100 °C for 12 hours, repeating this process three times. Then, solvent exchange was performed with anhydrous ethanol (100 mL), soaking for 12 hours each time, for a total of three times. Finally, the sample was activated at 150 °C and under high vacuum (<1 Pa) for 24 hours to obtain approximately 680 mg of white powdery biomimetic catalyst based on MOF material.

[0048] (3) The condensation reaction of glycine ethyl ester catalyzed by a biomimetic catalyst based on MOF material.

[0049] In a nitrogen-filled 100 mL Schlenk flask, glycine ethyl ester hydrochloride (279 mg, 2.0 mmol) and triethylamine (0.28 mL, 2.0 mmol) were added. Anhydrous tetrahydrofuran (THF, 20 mL) was added as a solvent, along with a MOF-based biomimetic catalyst (56 mg, 20% of the substrate mass). The reaction mixture was magnetically stirred in an oil bath at 60 °C for 48 hours.

[0050] After the reaction was complete, the solid catalyst was recovered by centrifugation (10,000 rpm, 5 minutes). The supernatant was evaporated under reduced pressure to remove the solvent, yielding an oily crude product. The crude product was purified by recrystallization: the oily crude product was dissolved in a minimal amount of hot ethyl acetate (approximately 5 mL), and n-hexane was slowly added dropwise to the hot solution until the solution began to become turbid. The flask was placed in a refrigerator at 4°C and allowed to stand overnight, resulting in the precipitation of white needle-like crystals. The crystals were collected by suction filtration through a Buchner funnel and washed with a small amount of cold hexane / ethyl acetate (5:1 v / v) mixed solvent. The crystals were dried under vacuum at room temperature for 12 hours to obtain pure glycyl-glycine ethyl ester (195 mg, yield 61%).

[0051] Example 3

[0052] (1) Preparation of IMTA ligands

[0053] In a 500 mL three-necked flask, 51.2 g (0.2 mol) of 2,5-dinitroterephthalic acid and 300 mL of methanol were added. Concentrated sulfuric acid (15 mL) was slowly added dropwise at room temperature, and the mixture was then refluxed at 75 °C for 36 hours. After the reaction was complete, the solution was cooled to room temperature, and a white solid precipitated. The solid was filtered, washed with ice-cold methanol, and then dried under vacuum at 60 °C to give dimethyl 2,5-dinitroterephthalate (54.8 g, 97% yield).

[0054] The product from the previous step (54.8 g) and 5.48 g of Pd / C were added to a 500 mL high-pressure reactor, along with 300 mL of methanol. The reactor was purged with nitrogen three times, and then hydrogen was introduced to a pressure of 0.8 MPa. The reaction was vigorously stirred at 60 °C for 24 hours. After the reaction was complete, the reactor was cooled and filtered to remove the catalyst. The filtrate was evaporated under reduced pressure to remove methanol, yielding a pale yellow solid, dimethyl 2,5-diaminoterephthalate (41.1 g, 96% yield).

[0055] Dimethyl 2,5-diaminoterephthalate (41.1 g, 0.18 mol), 4-(chloromethyl)-1H-imidazolium hydrochloride (28.8 g, 0.18 mol), and anhydrous potassium carbonate (75.9 g, 0.55 mol) were added to a 500 mL three-necked flask, along with 300 mL of anhydrous DMF. Under nitrogen protection, the mixture was heated to 90 °C and stirred for 36 hours. After the reaction was complete, the reaction mixture was poured into 1 L of water and extracted with ethyl acetate (3 × 300 mL). The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. After filtration, the solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol, v / v ratio from 100:1 to 50:1) to give a pure monosubstituted product (31.2 g, yield 56%).

[0056] The product from the previous step (31.2 g, 0.10 mol) was dissolved in 3M hydrochloric acid (200 mL) and cooled to -5°C. An aqueous solution of sodium nitrite (7.7 g, 0.11 mol) was slowly added dropwise, maintaining the temperature below 0°C, and the reaction was allowed to proceed for 3 hours. This diazonium salt solution was then slowly added dropwise to an aqueous solution of sodium hydrosulfide hydrate (NaSH·xH₂O, 22.8 g, approximately 0.41 mol) at 0°C (150 mL). After the addition was complete, the mixture was stirred at room temperature for 5 hours. The solution was neutralized to pH approximately 6 with 6M hydrochloric acid, then extracted with ethyl acetate, dried, and rotary evaporated to obtain a crude ester containing thiol groups. The crude ester was dissolved in methanol / water (2:1, 300 mL, v / v), and sodium hydroxide (24.4 g, 0.61 mol) was added. The mixture was refluxed at 80°C for 18 hours. After the reaction was complete, the mixture was cooled to room temperature. Under ice bath conditions, the pH was adjusted to 4.0 with 6M hydrochloric acid, resulting in the precipitation of a white precipitate. The precipitate was collected by vacuum filtration, washed three times with 500 mL of deionized water, and then washed once with 50 mL of ethanol. The obtained solid was dried under vacuum at 70 °C for 24 hours to obtain the final product IMTA (20.5 g, total yield of 68% for both steps).

[0057] (2) Synthesis of biomimetic catalysts based on MOF materials

[0058] In a 100 mL Lflon-lined reactor, zirconium tetrachloride (ZrCl4, 233 mg, 1.0 mmol), terephthalic acid (BDC, 166 mg, 1.0 mmol), and the functional ligand IMTA (57 mg, 0.2 mmol) were added. N,N-dimethylformamide (DMF, 50 mL) and acetic acid (5 mL) were also added. The mixture was sonicated for 30 minutes. The reactor was sealed and heated in an oven at 120 °C for 48 hours. After the reaction, the mixture was allowed to cool naturally. The product was collected by centrifugation and soaked in fresh DMF (50 mL) at 80 °C for 24 hours, repeated twice. Then, solvent exchange was performed with anhydrous ethanol (50 mL), soaking for 24 hours each time, for a total of two times. Finally, the sample was activated at 120 °C under high vacuum (<1 Pa) for 12 hours to obtain approximately 320 mg of a white powdery biomimetic catalyst based on MOF material.

[0059] (3) The condensation reaction of L-phenylalanine ethyl ester catalyzed by a biomimetic catalyst based on MOF material

[0060] In a nitrogen-filled 50 mL Schlenk flask, L-phenylalanine ethyl ester hydrochloride (459 mg, 2.0 mmol) and triethylamine (0.28 mL, 2.0 mmol) were added. Anhydrous dichloromethane (DCM, 20 mL) was added as a solvent, along with a MOF-based biomimetic catalyst (92 mg, 20% of the substrate mass). The reaction mixture was magnetically stirred in an oil bath at 40 °C for 72 hours. After the reaction was complete, the solid catalyst was recovered by centrifugation (12000 rpm, 3 min). The supernatant was evaporated under reduced pressure to remove the solvent, yielding an oily crude product. The crude product was purified by silica gel column chromatography: 5 g of 300-400 mesh silica gel was weighed and mixed with petroleum ether / ethyl acetate (volume ratio 10:1) to form a homogenate, which was then wet-packed into a 1.5 cm inner diameter chromatography column. The crude product was dissolved in 1 mL of dichloromethane, mixed with 80 mg of silica gel, evaporated to dryness, and then evenly spread on the top of the column. First, elute with petroleum ether / ethyl acetate (10:1, 100 mL, v / v), then gradually increase the petroleum ether / ethyl acetate ratio to 3:1. Monitor the eluent using a TLC plate (hexane / ethyl acetate 3:1 as the developing solvent). Combine the fractions containing the target product (L-phenylalanyl-L-phenylalanine ethyl ester), remove the solvent by rotary evaporation under reduced pressure, and give a pure white solid product (278 mg, 79% yield).

Claims

1. A biomimetic catalyst based on MOF materials, characterized in that, It comprises a zinc or zirconium metal node and a functional ligand containing a thiol, an imidazole, and a carboxyl group; said functional ligand is 2-((1H-imidazol-4-ylmethyl)amino)-5-mercaptoterephthalic acid or a derivative thereof.

2. A method for preparing a biomimetic catalyst based on MOF materials as described in claim 1, characterized in that, The process includes preparing functional ligands by dissolving metal salts, terephthalic acid, and functional ligands in a solvent in a specific ratio, adding a structure modifier, dispersing the mixture ultrasonically, and then carrying out a solvothermal reaction. After the reaction is complete, the product is collected by centrifugation, washed with organic solvent, exchanged with solvent, and activated under high temperature and vacuum to obtain a biomimetic catalyst based on MOF materials.

3. The method for preparing the biomimetic catalyst based on MOF material according to claim 2, characterized in that, The metal salt is zirconium tetrachloride (ZrCl4) or zinc nitrate (Zn(NO3)2).

4. The method for preparing the biomimetic catalyst based on MOF material according to claim 2, characterized in that, The molar ratio of the metal salt, terephthalic acid, and functional ligand is 1:(0.8-0.98):(0.02-0.2).

5. The method for preparing the biomimetic catalyst based on MOF material according to claim 2, characterized in that, The structure modifier is acetic acid or formic acid, and its addition amount is 1% to 10% of the total solvent volume.

6. The method for preparing the biomimetic catalyst based on MOF material according to claim 2, characterized in that, The solvothermal reaction is carried out at a temperature of 100-140℃ for 24-72 hours.

7. The method for preparing the biomimetic catalyst based on MOF material according to claim 2, characterized in that, The preparation method of 2-((1H-imidazol-4-ylmethyl)amino)-5-mercapto-terephthalic acid is as follows: 2,5-dinitro-terephthalic acid reacts with methanol under reflux catalysis of concentrated sulfuric acid to generate dimethyl 2,5-dinitro-terephthalate; the dinitro ester is hydrogenated and reduced to generate dimethyl 2,5-diamino-terephthalate; the diamino ester reacts with 4-(chloromethyl)-1H-imidazolium hydrochloride to introduce an imidazolium group, yielding a monosubstituted product; the monosubstituted product undergoes a diazotization reaction, followed by a reaction with sodium hydrosulfide to introduce a thiol group; the thiol-containing ester undergoes a hydrolysis reaction to generate 2-((1H-imidazol-4-ylmethyl)amino)-5-mercapto-terephthalic acid.

8. The application of the biomimetic catalyst based on MOF material as described in claim 1 in peptide synthesis, characterized in that, The application method is as follows: under the condition of no chemical coupling agent, a biomimetic catalyst based on MOF material is used to catalyze the condensation reaction of amino acid esters to generate peptide bonds.

9. The application of the MOF-based biomimetic catalyst in peptide synthesis according to claim 8, characterized in that, The catalytic reaction temperature is 30-60℃, and the reaction time is 12-48 hours; the amount of catalyst used is 5% to 20% of the initial mass of the amino acid ester.

10. The application of the MOF-based biomimetic catalyst according to claim 8 in peptide synthesis, characterized in that, After the catalytic reaction is completed, the catalyst is recovered by centrifugation or filtration. The recovered catalyst can be reused for the next catalytic reaction after washing and drying. The reaction solution is concentrated and purified by column chromatography or recrystallization to obtain the target peptide product.