Molecular tag DMDPM compound, preparation method thereof and application of molecular tag DMDPM compound in assisting homogeneous synthesis of heptapeptide H6K

Abstract

The invention relates to a molecular tag DMDPM compound, a preparation method thereof and application of the molecular tag DMDPM compound in assisting homogeneous synthesis of heptapeptide H6K, and belongs to the technical field of organic synthesis. The molecular tag DMDPM compound is 2, 4-dimethoxy-4 '-diphenylphosphinyl oxydiphenyl alcohol, and the molecular tag DMDPM compound is 2, 4-dimethoxy-4'-diphenylphosphinyl The method has the advantages of a liquid-phase synthesis method and a solid-phase synthesis method, H6K can be simply, quickly, economically and efficiently synthesized and prepared, and a 2, 4-dimethoxy-4 '-diphenylphosphinyloxy diphenyl alcohol small-molecule auxiliary group (also called as a carrier) can be recycled and directly reused, so that raw material waste is reduced, waste pollution is reduced, the cost is saved, and environmental protection is facilitated.

Description

Technical Field

[0001] This invention belongs to the field of organic synthesis technology, specifically relating to a molecularly labeled DMDPM compound, its preparation method, and its application in the assisted homogeneous synthesis of heptapeptide H6K. Background Technology

[0002] Polypeptide structures contain various coordinating amino acids, such as histidine, aspartic acid, and glutamic acid, which can covalently and adsorb onto metal ions to form peptide-metal chelates, such as those with Ca. 2+ Fe 2+ Zn 2+ Ni 2+ Coordination with metal ions. These peptide-metal chelates exhibit good thermal stability, combining the properties of both peptides and metal ions, and possess antibacterial and antioxidant properties. Furthermore, because short peptides are more easily transported and absorbed by the human intestine, peptide-metal chelates can be used as transport tags for metal elements in the food industry, improving the efficiency of metal element uptake by organisms. For example, peptide-zinc chelates can effectively supplement zinc ions, and their bioavailability is high. These peptide-metal chelates can not only be used for the transport of trace metal elements in the human body, but also for ion detection, fluorescent dye labeling, and protein purification. For instance, peptide structures containing aspartic acid and glutamic acid residues can form chelates with calcium ions. Introducing fluorescent groups onto these peptides can yield peptide fluorescent detection devices for calcium ion detection. Professor Keun-Hyeung Lee's research group modified histidine structures with pyrene sulfonyl chloride fluorophores. The imidazole group on histidine readily reacts with Zn... 2+ Coordination was performed, forming pyrene dimers, and a Zn-recognizing agent was designed and synthesized. 2+ A short peptide fluorescent chemical sensor.

[0003] At the same time, histidine also interacts with Ni 2+ There are strong interactions between the histidine and histidine, leading to the development of a histidine tag structure for the purification of recombinant proteins. The histidine tag is an amino acid motif composed of 6-10 histidines, with a very small molecular weight. It can be introduced into the N- or C-terminus of a recombinant protein to form a fusion protein without obscuring the epitopes and domains of other proteins within the fusion protein, nor affecting the protein's structure and function. Furthermore, the chelation of this histidine peptide with divalent metal ions (nickel, zinc, etc.) facilitates the purification of proteins using metal ion affinity chromatography (IMAC).

[0004] The specific purification principle is to utilize the stationary phase medium and divalent metal cations (Ni) 2+ Chelation occurs, resulting in a large amount of Ni on the adsorption column. 2+At higher pH levels, the His-tag short peptide carries a negative charge and forms coordination bonds with metal ions, becoming immobilized on the chromatography medium. Therefore, His-tag proteins selectively bind to the metal-equipped chromatography medium, while other impurity proteins cannot bind or only weakly bind, thus separating the fusion protein from other proteins. Finally, competitive elution is performed using imidazole, or the pH is adjusted to 5.9 to protonate histidine, preventing it from binding to Ni. 2+ Due to electrostatic interaction, it is eluted, yielding a high-purity His-tag protein. The specific separation process is as follows: Figure 1 .

[0005] IMAC is currently the most commonly used method for His-tag protein purification in prokaryotic protein expression and purification. 2+ Ni is most widely used in affinity purification; depending on the binding group, it... 2+ They can be divided into Ni-NTA and Ni-IDA. Ni-NTA chelates tetravalent proteins, while Ni-IDA chelates trivalent proteins, resulting in higher loading capacity. Their binding structures with His-tag proteins are as follows: Figure 2 .

[0006] In addition, a modified His tag consisting of six His residues and one Cys residue (CH6-tag) has been reported. The thiol group of the Cys residue can covalently bind to some acceptors, such as maleimide and chloroacetyl groups.

[0007] His-tags are widely used not only for affinity purification in metal affinity chromatography, but also in research methods such as immunoblotting, fluorescent dye labeling, refolding studies, and viral tag development. His-tags offer advantages such as small molecular weight, elution under mild conditions, and high purity. Currently, no methods for the chemical synthesis of H6K molecular tags have been reported in existing technologies. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a molecularly tagged DMDPM compound, its preparation method, and its application in the assisted homogeneous synthesis of heptapeptide H6K. This invention overcomes many defects in existing synthetic processes by introducing small molecule tagging technology. Compared to traditional chemical synthesis methods, this method significantly simplifies the complex homogeneous reaction process, reduces separation and purification steps, and shortens the production cycle. Compared to solid-phase synthesis methods, this technology overcomes production scale limitations, significantly reduces the consumption of expensive raw materials, and effectively reduces the generation of resin waste. While increasing production capacity, it achieves a more environmentally friendly and economical production process, providing a practical solution for the large-scale preparation of peptide H6K.

[0009] The primary objective of this invention is to provide a molecularly labeled DMDPM compound, with the structural formula shown below: ; Among them, the DMDPM compound is 2,4-dimethoxy-4'-diphenylphosphonodiphenylethanol.

[0010] The second objective of this invention is to provide a method for preparing a molecularly labeled DMDPM compound, comprising the following steps: 2,4-Dimethoxy-4'-hydroxybenzophenone was dissolved in tetrahydrofuran, sealed, placed in an ice bath, and then triethylamine was added. After mixing well, diphenylphosphine chloride diluted with tetrahydrofuran was added. After the reaction was completed, a white solid product was obtained. The white solid product was dissolved in a mixed solution of methanol and tetrahydrofuran. The mixed solution was placed in an ice bath, and NaBH4 was added in several portions. The mixture was then sealed and stirred under ice bath conditions. After the reaction was completed, the mixture was quenched with saturated NH4Cl solution. The precipitate was removed by filtration. The filtrate was concentrated under reduced pressure and washed to obtain the DMDPM compound.

[0011] The third objective of this invention is to provide an application of the molecularly labeled DMDPM compound in the homogeneous synthesis of heptapeptide H6K.

[0012] The fourth objective of this invention is to provide a method for homogeneous synthesis of heptapeptide H6K assisted by a molecularly labeled DMDPM compound, comprising the following steps: Using DMDPM compound as an auxiliary group, it is reacted with amino acid under the action of a dehydrating coupling agent, so that the C-terminus of the amino acid is connected to the auxiliary group to generate compound A; wherein the amino acid is L-lysine Fmoc-Lys(Boc)-OH with both amino groups protected. The N-terminal Fmoc of compound A was deprotected to obtain deprotected product B; Fmoc-His(Trt)-OH, EDCI, HOBt and DIEA were dissolved in dichloromethane, and then deprotected product B was added to carry out a coupling reaction to obtain compound C. Fmoc was removed from compound C to obtain deprotected product D; Fmoc-His(Trt)-OH, EDCI, HOBt, and DIEA were dissolved in dichloromethane, and then deprotected product D was added to carry out a coupling reaction. After the reaction was completed, Fmoc was removed to obtain deprotected product E. Subsequently, this step was repeated, and deprotected product D was replaced by deprotected product E to continue coupling four Fmoc-His(Trt)-OH to obtain the compound H-His(Trt)-His(Trt)-His(Trt)-His(Trt)-His(Trt)-Lys(Boc)-DMDPM. The molecular tag DMDPM on the compound H-His(Trt)-His(Trt)-His(Trt)-His(Trt)-His(Trt)-His(Trt)-Lys(Boc)-DMDPM was cleaved to obtain the heptapeptide H6K.

[0013] The fifth objective of this invention is to provide a heptapeptide H6K, the structural formula of which is as follows: .

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a molecularly tagged DMDPM compound, its preparation method, and its application in the assisted homogeneous synthesis of heptapeptide H6K. By introducing small molecule tagging technology, this invention overcomes many shortcomings of existing synthetic processes. Compared to traditional chemical synthesis methods, this method significantly simplifies the complex homogeneous reaction process, reduces separation and purification steps, and shortens the production cycle. Compared to solid-phase synthesis methods, this technology overcomes production scale limitations, significantly reduces the consumption of expensive raw materials, and effectively reduces the generation of resin waste. While increasing production capacity, it achieves a more environmentally friendly and economical production process, providing a practical solution for the large-scale preparation of peptide H6K.

[0015] This invention combines the advantages of liquid-phase and solid-phase synthesis methods, enabling the simple, rapid, economical, and efficient synthesis and preparation of H6K. Furthermore, the small molecule auxiliary group (also known as the carrier) of 2,4-dimethoxy-4'-diphenylphosphonodiphenylmethanol can be recycled and directly reused, reducing raw material waste, minimizing waste pollution, saving costs, and benefiting the environment. Attached Figure Description

[0016] Figure 1 The principle of Ni-NTA chromatography column for fusion protein purification; Figure 2 This diagram illustrates the binding of Ni-IDA and Ni-NTA to the fusion protein. Figure 3 The results are from the HPLC analysis of H6K. Detailed Implementation

[0017] The present invention will be further described below with reference to specific embodiments. However, it should be understood that the listed embodiments are only for the purpose of understanding the core methods and application fields of the present invention, but the scope of the present invention is not limited thereto.

[0018] Unless otherwise specified, the experimental and detection methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0019] The purpose of this invention is to provide a novel method for synthesizing heptapeptide structures with a histidine tag of H6K (HHHHHHK) assisted by DMDPM tagging. By introducing small molecule tagging technology, this method overcomes many defects in existing synthesis processes.

[0020] To achieve the above objectives, the first aspect of the present invention provides a molecularly labeled DMDPM compound with the following structural formula: ; Among them, the DMDPM compound is 2,4-dimethoxy-4'-diphenylphosphonodiphenylethanol.

[0021] A second aspect of the present invention provides a method for preparing the molecularly labeled DMDPM compound of claim 1, comprising the following steps: 2,4-Dimethoxy-4'-hydroxybenzophenone was dissolved in tetrahydrofuran, sealed, placed in an ice bath, and then triethylamine was added. After mixing well, diphenylphosphine chloride diluted with tetrahydrofuran was added. After the reaction was completed, a white solid product was obtained. The white solid product was dissolved in a mixed solution of methanol and tetrahydrofuran. The mixed solution was placed in an ice bath, and NaBH4 was added in several portions. The mixture was then sealed and stirred under ice bath conditions. After the reaction was completed, the mixture was quenched with saturated NH4Cl solution. The precipitate was removed by filtration. The filtrate was concentrated under reduced pressure and washed to obtain the DMDPM compound.

[0022] An exemplary method for preparing a 2,4-dimethoxy-4'-diphenylphosphonoyloxydibenzyl alcohol compound includes the following steps: 1) Accurately weigh 2,4-dimethoxy-4'-hydroxybenzophenone (2.58 g, 10.0 mmol, 1.0 eq) and place it in a flask. Add 40 mL of tetrahydrofuran to dissolve it. Seal the flask with a balloon and stir in an ice bath for 10 min to lower the system temperature to a minimum. Slowly add triethylamine (1.67 mL, 12 mmol, 1.2 eq) to the solution and stir in an ice bath for 20 min. Weigh diphenylphosphine chloride (2.46 g, 10.4 mmol, 1.04 eq), dilute it with tetrahydrofuran, and slowly add it dropwise to the reaction system in multiple portions. White fumes are generated at the mouth of the flask, and the reaction solution becomes turbid. Monitor the reaction progress by TLC (developing solvent). V DCM : V MeOHThe reaction mixture was 20:1. After approximately 2 hours, the reactants were completely reacted, and the reaction was stopped. The reaction solution was filtered, concentrated under reduced pressure to remove the solvent, and the crude product was dissolved in 50 mL of ethyl acetate. It was washed three times with 40 mL of saturated NaHCO3 solution, and the organic phase was dried over anhydrous Na2SO4. The solvent was then removed by concentration under reduced pressure. The product was dried under vacuum to obtain a white solid.

[0023] 2) Accurately weigh the product from step 1) (2.3 g, 5 mmol, 1.0 eq) and dissolve it in a 20 mL mixture of methanol and tetrahydrofuran. Place the mixture in an ice bath. Weigh out NaBH4 (227 mg, 6 mmol, 1.2 eq) and add it to the reaction solution in several portions. Seal the reaction flask with a balloon and stir the mixture in an ice bath for 3 h. Monitor the reaction progress using a TCL (developing solvent). V DCM : V MeOH (20:1 ratio) After the reactants reacted completely, 5 mL of saturated NH4Cl solution was added to quench the reaction, and the precipitate was removed by filtration. The filtrate was concentrated under reduced pressure to remove the solvent, dissolved in ethyl acetate, washed with distilled water and saturated brine, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The product was dried under vacuum to obtain a white, foamy solid, which is a compound of the 2,4-dimethoxy-4'-diphenylphosphonodibenzyl alcohol class (DMDPM).

[0024] A third aspect of this invention provides the application of a molecularly labeled DMDPM compound in the homogeneous synthesis of heptapeptide H6K.

[0025] A fourth aspect of this invention provides a method for homogeneous synthesis of heptapeptide H6K assisted by a molecularly tagged DMDPM compound, comprising the following steps: Using DMDPM compound as an auxiliary group, it is reacted with amino acid under the action of a dehydrating coupling agent, so that the C-terminus of the amino acid is connected to the auxiliary group to generate compound A (Fmoc-Lys(Boc)-DMDPM); wherein the amino acid is L-lysine Fmoc-Lys(Boc)-OH with both amino groups protected. The N-terminal Fmoc of compound A was removed to obtain deprotected product B (H-Lys(Boc)-DMDPM). Fmoc-His(Trt)-OH, EDCI, HOBt, and DIEA were dissolved in dichloromethane, and then deprotected product B was added to carry out a coupling reaction to obtain compound C (Fmoc-His(Trt)-Lys(Boc)-DMDPM). Fmoc was removed from compound C to obtain deprotected product D (H-His(Trt)-Lys(Boc)-DMDPM). Fmoc-His(Trt)-OH, EDCI, HOBt, and DIEA were dissolved in dichloromethane, and then deprotected product D was added for coupling reaction. After the reaction was completed, Fmoc was removed to obtain deprotected product E (H-His(Trt)-His(Trt)-Lys(Boc)-DMDPM). Subsequently, this step was repeated, and deprotected product D was replaced by deprotected product E to continue coupling four Fmoc-His(Trt)-OH to obtain the compound H-His(Trt)-His(Trt)-His(Trt)-His(Trt)-His(Trt)-His(Trt)-Lys(Boc)-DMDPM. Specifically, based on the deprotected product E (H-His(Trt)-His(Trt)-Lys(Boc)-DMDPM), the first Fmoc-His(Trt)-OH is coupled again, and then Fmoc is removed to obtain H-His(Trt)-His(Trt)-His(Trt)-Lys(Boc)-DMDPM; the second Fmoc-His(Trt)-OH is coupled again, and then Fmoc is removed to obtain H-His(Trt)-His(Trt)-His(Trt)-His(Trt)-Lys(Boc)-DMDPM; the third Fmoc-His(Trt)-OH is coupled again, and then Fmoc is removed to obtain H-His(Trt)-His(Trt)-His(Trt)-His(Trt)-Lys(Boc)-DMDPM. Continue coupling with the fourth Fmoc-His(Trt)-OH, then remove Fmoc to obtain H-His(Trt)-His(Trt)-His(Trt)-His(Trt)-His(Trt)-His(Trt)-Lys(Boc)-DMDPM (HHHHHHK-DMDPM).

[0026] The molecular tag DMDPM on the compound H-His(Trt)-His(Trt)-His(Trt)-His(Trt)-His(Trt)-His(Trt)-Lys(Boc)-DMDPM was cleaved to obtain the heptapeptide H6K.

[0027] The Fmoc extraction process includes: The Fmoc-protected compound was dissolved in acetonitrile, and then diethylamine was added to react. After the reaction was completed, the acetonitrile and diethylamine were removed by vacuum concentration at room temperature. DCM was added repeatedly for vacuum concentration to remove diethylamine to the greatest extent. After the solvent was evaporated to dryness, ethyl acetate was added to dissolve the compound. Petroleum ether was then added dropwise while shaking until no target product was found in the supernatant. The solid and liquid phases were separated by centrifugation and washed until no impurities were found in the supernatant to obtain the deprotected product.

[0028] The dehydrating coupling agent is a 1:1 molar ratio of a dehydrating coupling activator and an alkaline substance; wherein the dehydrating coupling activator is EDCI, and the alkaline substance is DIEA or DMAP. The molar ratio of the amino acid to the auxiliary group is 1 to 1.1.

[0029] The shearing process includes: The compound H-His(Trt)-His(Trt)-His(Trt)-His(Trt)-His(Trt)-Lys(Boc)-DMDPM was added to a TFA / TIS / H2O mixture and stirred at room temperature. After the reaction was complete, dichloromethane was added to the resulting mixture, and TFA, H2O, and TIS were removed by rotary evaporation at 40°C. This process was repeated with multiple additions of dichloromethane. After rotary drying, cold diethyl ether was added, and the mixture was sonicated. The precipitate was then pulverized by ultrasonic vibration and centrifuged to obtain the cleaved crude peptide. Cold diethyl ether was added again, and the mixture was washed with ultrasonic waves approximately three times or more until the supernatant was free of impurities. After centrifugation, the cleaved heptapeptide H6K was obtained. The ether phase was then concentrated and purified by column chromatography to obtain the cleaved DMDPM tag. The volume ratio of TFA / TIS / H2O was 95:2.5:2.5.

[0030] An exemplary method for homogeneous synthesis of heptapeptide H6K assisted by a molecularly labeled DMDPM compound includes the following steps: 1) Coupling of auxiliary groups The auxiliary group is 2,4-dimethoxy-4'-diphenylphosphonodiphenylethanol; the amino acid is Fmoc-Lys(Boc)-OH; The auxiliary group is used to replace the resin in solid-phase peptide synthesis. The amino acid is stirred and reacted with the amino acid under the action of a dehydrating coupling agent, so that the C-terminus of the amino group Fmoc-Lys(Boc)-OH is connected to the auxiliary group to generate compound A, Fmoc-Lys(Boc)-DMDPM. In step 1), the reaction is stirred at 0°C for 1 to 3 hours; the molar ratio of the amino acid to the auxiliary group is 1 to 1.1; the dehydration coupling agent is a 1:1 molar ratio of dehydration coupling activator and alkaline substance; wherein, the dehydration coupling activator is EDCI, and the alkaline substance is DIEA or DMAP. 2) Separation and purification Add 60 mL of ethyl acetate to compound A obtained in step 1) to dissolve it, wash it three times with 50 mL of saturated ammonium chloride aqueous solution, saturated sodium bicarbonate solution and saturated brine respectively, dry the organic phase with anhydrous sodium sulfate, evaporate the solvent to obtain white foam; Weigh Fmoc-Lys(Boc)-DMDPM(4-1) (4.2 g, 4.6 mmol, 1 eq) into a round-bottom flask, dissolve in 18 mL of acetonitrile, and add 6 mL of diethylamine (V MeCN / V DEA =3:1), TCL monitoring throughout (developing solvent DCM:MeOH=20:1), until the reactants reacted completely, approximately 2 hours. After the reaction, the mixture was concentrated under reduced pressure at room temperature to remove acetonitrile and diethylamine, with dichloromethane added multiple times for further concentration under reduced pressure to remove diethylamine to the greatest extent. After the solvent was evaporated to dryness, 6 mL of ethyl acetate was added to dissolve the product, and then petroleum ether was added while shaking. A white solid appeared, and petroleum ether was added dropwise until no target product was found in the supernatant. A total of 60 mL of petroleum ether (V) was added. PE :V EA =10:1), centrifuge to separate the solid and liquid phases, wash until the supernatant is free of impurities, and obtain the deprotected product, a white foamy solid of 2.92 g, with a yield of 92%.

[0031] 3) Remove N-terminal Fmoc Dissolve 6 mL of diethylamine in 18 mL of acetonitrile (V) MeCN / VDEA =3:1) Reagent dissolution step 2) The purified compound A was stirred and reacted at room temperature for 0.5 to 2 hours; after the reaction was completed, the mixture was concentrated under reduced pressure at room temperature to remove acetonitrile and diethylamine, and dichloromethane was added repeatedly for further concentration under reduced pressure to remove diethylamine to the greatest extent. After the solvent was evaporated to dryness, 6 mL of ethyl acetate was added to dissolve it, and then petroleum ether was added while shaking. A white solid appeared. Petroleum ether was added dropwise until no target product was found in the supernatant. A total of 60 mL of petroleum ether (V) was added. PE :V EA =10:1), centrifuge to separate the solid and liquid phases, wash until the supernatant is free of impurities, and obtain the deprotected product B.

[0032] 4) Peptide coupling Experimental synthesis steps of H-His(Trt)-Lys(Boc)-DMDPM: Accurately weigh Fmoc-His(Trt)-OH (2.79 g, 4.51 mmol, 1.1 eq), EDCI (861 mg, 4.51 mmol, 1.1 eq), HOBt (609 mg, 4.51 mmol, 1.1 eq), and DIEA (802 µL, 4.51 mmol, 1.1 eq), dissolve in 25 mL of dichloromethane, stir at 0 °C for 0.5 h to activate the amino acids, then add the tag (2.82 g, 4.10 mmol, 1 eq). Monitor the reaction progress by TLC (developing solvent DCM:MeOH = 20:1). After approximately 3 h of reaction, the starting materials are completely reacted. Stop the reaction, evaporate the solvent, add 60 mL of ethyl acetate to dissolve, and then use 50 mL of... The organic phase was washed three times with saturated ammonium chloride aqueous solution, saturated sodium bicarbonate solution, and saturated brine. The organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain a white, foamy solid C. Fmoc-His(Trt)-Lys(Boc)-DMDPM (4-5) was dissolved in 18 mL of acetonitrile, and 6 mL of diethylamine (V...) was added. MeCN / V DEA =3:1), TCL monitoring throughout (developing solvent DCM:MeOH=20:1), until the reactants reacted completely, approximately 2 hours. After the reaction, the mixture was concentrated under reduced pressure at room temperature to remove acetonitrile and diethylamine, with DCM added repeatedly for further concentration under reduced pressure to remove diethylamine to the greatest extent possible. After the solvent was evaporated to dryness, 6 mL of ethyl acetate was added to dissolve the product. Then, petroleum ether was added while shaking, resulting in the appearance of a white solid. Petroleum ether was added dropwise until no target product was found in the supernatant. A total of 48 mL of petroleum ether (V) was added. PE :V EA =8:1), centrifuge to separate the solid and liquid phases, wash until the supernatant is free of impurities, and obtain the deprotected product D, a white foamy solid.

[0033] Experimental synthesis steps of H-His(Trt)-His(Trt)-Lys(Boc)-DMDPM: Accurately weigh Fmoc-His(Trt)-OH (1.80 g, 2.90 mmol, 1.1 eq), EDCI (554 mg, 2.90 mmol, 1.1 eq), HOBt (392 mg, 2.90 mmol, 1.1 eq), and DIEA (516 µL, 2.90 mmol, 1.1 eq), dissolve in 25 mL dichloromethane, stir at 0 °C for 0.5 h to activate the amino acids, then add compound D (3.4 g, 2.64 mmol, 1 eq). Monitor the reaction progress by TLC (evolving solvent DCM:MeOH = 20:1). After approximately 3 h of reaction, the starting materials are completely reacted. Stop the reaction, evaporate the solvent, and directly proceed to the next step. Dissolve the crude product in 18 mL acetonitrile, add 6 mL diethylamine (V... MeCN / V DEA =3:1), TCL monitoring throughout (developing solvent DCM:MeOH=10:1), until the reactants reacted completely, approximately 2 hours. After the reaction, the mixture was concentrated under reduced pressure at room temperature to remove acetonitrile and diethylamine, with dichloromethane added multiple times for further concentration under reduced pressure to remove diethylamine to the greatest extent. 50 mL of dichloromethane was added to dissolve the mixture, and it was washed three times with 40 mL of saturated sodium bicarbonate solution and saturated brine, respectively. The organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated. After the solvent was evaporated to dryness, 6 mL of ethyl acetate was added to dissolve the mixture. Then, petroleum ether was added while shaking, resulting in the appearance of a white solid. Petroleum ether was added dropwise until no target product was found in the supernatant. A total of 48 mL of petroleum ether (V) was added. PE :V EA =8:1), centrifuge to separate the solid and liquid phases, wash until there are no impurities in the supernatant, and obtain the deprotected product E, a white foamy solid.

[0034] Subsequent peptide synthesis protocols repeat the above steps, with amino acids, EDCI, HOBt, and DIEA being coupled to four Fmoc-His(Trt)-OH groups in a 1.1 equivalence excess ratio.

[0035] This section utilizes the DMDPM tag to obtain the HHHHHHK-DMDPM structure in high yield, achieving the synthesis of H6K short peptides and verifying the feasibility and stability of the DMDPM tag in peptide synthesis.

[0036] 5) Synthetic schemes involving cleavage of peptide chains from tags Weigh 500 mg (0.17 mmol) of compound HHHHHHK-DMDPM into a 10 mL TFA / TIS / H2O (v / v / v, 95:2.5:2.5) mixture, stir at room temperature, and monitor the reaction progress by TLC. After 3 h, the starting material was completely reacted. Add dichloromethane to the mixture, and remove TFA, H2O, and TIS by rotary evaporation at 40 °C. Repeat the above operation by adding dichloromethane several times. After rotary drying, add cold diethyl ether, sonicate, and a pale yellow solid appears. Crush the precipitate by ultrasonic vibration, centrifuge to obtain the cleaved crude peptide, continue adding cold diethyl ether and ultrasonic washing, washing about three times or more until the supernatant is free of impurities, and centrifuge to obtain the cleaved peptide pale yellow solid. Concentrate the ether phase, purify by column chromatography, and obtain the cleaved DMDPM tag. The side chain deprotection reaction conditions are: stirring at room temperature for 1-2 hours.

[0037] See the following synthetic route for heptapeptide H6K: .

[0038] The molecular structure of the deprotected product B (H-Lys(Boc)-DMDPM) is as follows: .

[0039] The molecular structure of Fmoc-Lys(Boc)-DMDPM is:

[0040] The molecular structure of the deprotected product D (H-His(Trt)-Lys(Boc)-DMDPM) is as follows: .

[0041] The molecular structure of Fmoc-His(Trt)-Lys(Boc)-DMDPM is: ; The molecular structure of Fmoc-His(Trt)-His(Trt)-Lys(Boc)-DMDPM is: ; The molecular structure of H-His(Trt)-His(Trt)-Lys(Boc)-DMDPM is: ; The general molecular structural formula of H-His(Trt)-His(Trt)-His(Trt)-Lys(Boc)-DMDPM is: ; The general molecular formula of H-His(Trt)-His(Trt)-His(Trt)-His(Trt)-Lys(Boc)-DMDPM is: ; The general molecular formula of H-His(Trt)-His(Trt)-His(Trt)-His(Trt)-Lys(Boc)-DMDPM is: ; The molecular structural formula of the H-His(Trt)-His(Trt)-His(Trt)-His(Trt)-His(Trt)-His(Trt)-Lys(Boc)-DMDPM compound is as follows: .

[0042] The fifth aspect of this invention provides a heptapeptide H6K, the structural formula of which is as follows: .

[0043] Based on the significance and wide application of the His-tag structure, this invention proposes a method for synthesizing H6K (HHHHHHK) using tag-assisted synthesis. Referring to the structure of H6K, a lysine residue is introduced at the carboxyl terminus of the His-tag to obtain a heptapeptide structure. The lysine residue has an amino group on its side, which can be linked to other groups for modification.

[0044] In this invention, some commonly used abbreviations have the following meanings: Boc: tert-Butoxycarbonyl; DCM: dichloromethane CH2Cl2; DEA: diethylamine; DMAP: 4-dimethylaminopyridine; DMDPM: 2,4-Dimethoxy-4'-diphenylphosphonodiphenylethanol; Fmoc: fluorenemethoxycarbonyl; DIEA: diisopropylethylamine; HOBt: 1-hydroxybenzotriazole; TFA: trifluoroacetic acid; THF: tetrahydrofuran.

[0045] The following examples provide specific synthetic methods for preparing the above-mentioned compounds and their corresponding intermediate compounds.

[0046] Example 1 In this embodiment, a high-purity heptapeptide H6K was synthesized using 2,4-dimethoxy-4'-diphenylphosphonodiphenylethanol as an auxiliary support. The synthetic route of the 2,4-dimethoxy-4'-diphenylphosphonodiphenylethanol support is as follows: ; The synthesis steps of 2,4-dimethoxy-4'-diphenylphosphinobenzophenone: Accurately weigh 2,4-dimethoxy-4'-hydroxybenzophenone (2.58 g, 10.0 mmol, 1.0 eq) and place it in a flask. Add 40 mL of tetrahydrofuran to dissolve it. Seal the flask with a balloon and stir in an ice bath for 10 min to lower the system temperature to a minimum. Slowly add triethylamine (1.67 mL, 12 mmol, 1.2 eq) to the solution and stir in an ice bath for 20 min. Weigh 2.46 g (10.4 mmol, 1.04 eq), dilute it with tetrahydrofuran, and slowly add it dropwise to the reaction system in multiple portions. White fumes are generated at the mouth of the flask, and the reaction solution becomes turbid. Monitor the reaction progress by TLC (developing solvent). V DCM : V MeOH The reaction mixture was prepared at a ratio of 20:1. After approximately 2 hours, the reactants were completely reacted, and the reaction was stopped. The reaction solution was filtered, concentrated under reduced pressure to remove the solvent, and the crude product was dissolved in 50 mL of ethyl acetate. It was washed three times with 40 mL of saturated NaHCO3 solution, and the organic phase was dried over anhydrous Na2SO4. The solvent was then removed by concentration under reduced pressure. The product was dried under vacuum to obtain a white solid.

[0047] Structural characterization data of 2,4-dimethoxy-4'-diphenylphosphonobenzophenone: 1H NMR (400MHz, DMSO- d6 ) δ 7.98 – 7.88 (m, 4H), 7.69 – 7.52 (m, 8H), 7.39 (d, J = 8.7 Hz, 2H), 7.29 (d, J = 8.4 Hz, 1H), 6.71 – 6.59 (m, 2H), 3.84 (s, 3H), 3.58 (s, 3H).13C NMR (101 MHz, DMSO- d6 ) δ 193.88, 163.53, 159.31, 154.52 (d, J = 8.1 Hz), 135.16, 133.41 (d, J = 2.9 Hz), 131.99 (d, J = 10.6 Hz), 131.67, 130.86 (d, J =136.4 Hz), 129.47 (d, J = 13.2 Hz), 121.06, 120.77 (d, J= 5.5 Hz), 106.08,99.21, 56.00, 55.97. 31P NMR (162 MHz, DMSO- d6 ) δ 29.97. HRMS (ESI) m / z calcdfor C 27 H 23 O5PNa + (M+Na) + 481.11753, found 481.11761. Experimental synthesis of 2,4-dimethoxy-4'-diphenylphosphonobenzophenone (DMDPM): Accurately weigh 2,4-dimethoxy-4'-diphenylphosphonobenzophenone (2.3 g, 5 mmol, 1.0 eq) from the previous step and dissolve it in a 20 mL mixture of methanol and tetrahydrofuran. Place the mixture in an ice bath. Weigh 227 mg of NaBH4 (6 mmol, 1.2 eq) and add it to the reaction solution in several portions. Seal the reaction flask with a balloon and stir the mixture in an ice bath for 3 h. Monitor the reaction progress using a TCL (developing solvent). V DCM : V MeOH (20:1 ratio) After the reactants reacted completely, 5 mL of saturated NH4Cl solution was added to quench the reaction, and the precipitate was removed by filtration. The filtrate was concentrated under reduced pressure to remove the solvent, dissolved in ethyl acetate, washed with distilled water and saturated brine, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The product was then dried under vacuum to obtain a white, foamy solid.

[0048] Structural characterization data of 2,4-dimethoxy-4'-diphenylphosphonodiphenylethanol: 1H NMR (400 MHz, DMSO- d6 ) δ 7.89 (dd, J = 12.3, 7.5 Hz, 4H), 7.65 – 7.53 (m, 6H), 7.30 – 7.12(m, 5H), 6.50 (s, 24 2H), 5.82 (d, J = 4.5 Hz, 1H), 5.77 (s, 1H), 3.70 (d, J =10.8 Hz, 6H). 13C NMR (126 MHz, DMSO- d6 ) δ 159.90, 156.96, 149.51 (d, J = 8.3Hz), 142.62, 133.16 (d, J= 2.8 Hz), 131.95 (d, J = 10.6 Hz), 130.86(d, J = 136.2Hz), 129.37 (d, J = 13.3 Hz), 128.05, 127.61, 126.23, 120.36 (d, J = 4.6 Hz),105.24, 98.55, 67.59, 55.86, 55.58. 31P NMR (162 MHz, DMSO- d6 ) δ 28.89. HRMS(ESI) m / z calcd for C 27 H 25 O5PNa + (M+Na) + 483.13318, found 483.13354. Experimental synthesis steps of Fmoc-Lys(Boc)-DMDPM: Accurately weigh Fmoc-Lys(Boc)-OH (3.38 g, 7.6 mmol, 1.2 eq), EDCI (1.38 g, 7.2 mmol, 1.2 eq), and DMAP (88 mg, 0.72 mmol, 0.12 eq), dissolve in 50 mL of dichloromethane, and stir at 0 °C for 0.5 h to activate the amino acids. Then add the labeled DMDPM (2.76 g, 6 mmol, 1 eq). Monitor the reaction progress by TLC (evolving solvent DCM:MeOH = 20:1). After about 3 h of reaction, the reactants were completely reacted, and the reaction was stopped. Evaporate the solvent, add 60 mL of ethyl acetate to dissolve, and wash three times with 50 mL of saturated ammonium chloride aqueous solution, saturated sodium bicarbonate solution, and saturated brine, respectively. Dry the organic phase with anhydrous sodium sulfate, evaporate the solvent, and obtain a white foamy solid.

[0049] Structural characterization data of Fmoc-Lys(Boc)-DMDPM: 1 H NMR (400 MHz, DMSO- d 6) δ 7.86(t, J = 10.6 Hz, 6H), 7.77 – 7.68 (m, 2H), 7.63 – 7.47 (m, 6H), 7.41 (d, J = 7.6Hz, 3H), 7.32 (d, J = 7.2 Hz, 2H), 7.19 (d, J= 7.6 Hz, 3H), 7.11 (d, J = 8.4 Hz, 2H), 6.90 (d, J = 6.8 Hz, 2H), 6.55 – 6.40 (m, 2H), 4.30 – 4.16 (m, 3H), 4.10 –3.98 (m, 1H), 3.69 (d, J = 20.8 Hz, 6H), 2.86 (s, 2H), 1.63 (d, J = 30.8 Hz, 2H), 1.37 (s, 13H). 13 C NMR (126 MHz, DMSO- d 6) δ 160.87, 157.11, 150.41, 144.29,141.21, 137.69, 137.06, 133.22, 131.88, 131.84, 130.75, 129.46, 129.36,128.46, 128.14, 127.76, 127.54, 124.78, 121.86, 120.74, 120.60, 120.50,105.41, 98.46, 78.82, 71.11, 66.21, 60.32, 56.07, 55.59, 47.09, 40.59, 31.15, 29.09, 28.74, 23.20. 31 P NMR (162 MHz, DMSO- d 6) δ 29.20. Weigh Fmoc-Lys(Boc)-DMDPM (4.2 g, 4.6 mmol, 1 eq) into a round-bottom flask, dissolve in 18 mL of acetonitrile, and add 6 mL of diethylamine (V MeCN / V DEA =3:1), TCL monitoring throughout (developing solvent DCM:MeOH=20:1), until the reactants reacted completely, approximately 2 hours. After the reaction, the mixture was concentrated under reduced pressure at room temperature to remove acetonitrile and diethylamine, with dichloromethane added multiple times for further concentration under reduced pressure to remove diethylamine to the greatest extent. After the solvent was evaporated to dryness, 6 mL of ethyl acetate was added to dissolve the product, and then petroleum ether was added while shaking. A white solid appeared, and petroleum ether was added dropwise until no target product was found in the supernatant. A total of 60 mL of petroleum ether (V) was added. PE :V EA =10:1), centrifuge to separate the solid and liquid phases, wash until there are no impurities in the supernatant, and obtain the deprotected product, a white foamy solid.

[0050] Structural characterization data of H-Lys(Boc)-DMDPM: 1 H NMR (400 MHz, DMSO- d 6) δ 7.88 (s, 4H), 7.60 (s, 2H), 7.55 (s, 4H), 7.20 (s, 5H), 6.90 (s, 1H), 6.79 (s, 1H), 6.49 (s, 2H), 3.73 (d, J = 6.1 Hz, 6H), 3.29 (s, 1H), 2.83 (s, 2H), 1.37 (s, 15H). 13 C NMR (126 MHz, DMSO- d 6) δ 174.35, 161.80, 156.86, 155.95, 133.25,131.96, 131.88, 131.83, 130.74, 129.48, 129.35, 128.59, 128.50, 128.04,120.83, 105.50, 99.40, 77.78, 69.47, 56.77, 56.12, 55.69, 54.60, 54.42,40.26, 33.63, 29.36, 28.75, 22.85. 31 P NMR (162 MHz, DMSO- d 6) δ 29.29. Experimental synthesis steps of H-His(Trt)-Lys(Boc)-DMDPM: Accurately weigh Fmoc-His(Trt)-OH (2.79 g, 4.51 mmol, 1.1 eq), EDCI (861 mg, 4.51 mmol, 1.1 eq), HOBt (609 mg, 4.51 mmol, 1.1 eq), and DIEA (802 µL, 4.51 mmol, 1.1 eq), dissolve in 25 mL of dichloromethane, stir at 0 °C for 0.5 h to activate the amino acids, then add the tag (2.82 g, 4.10 mmol, 1 eq). Monitor the reaction progress by TLC (developing solvent DCM:MeOH = 20:1). After approximately 3 h of reaction, the starting materials are completely reacted. Stop the reaction, evaporate the solvent, add 60 mL of ethyl acetate to dissolve, and then use 50 mL of... The organic phase was washed three times with saturated ammonium chloride aqueous solution, saturated sodium bicarbonate solution, and saturated brine. The organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain a white, foamy solid. Fmoc-His(Trt)-Lys(Boc)-DMDPM was dissolved in 18 mL of acetonitrile, and 6 mL of diethylamine (V...) was added. MeCN / V DEA =3:1), TCL monitoring throughout (developing solvent DCM:MeOH=20:1), until the reactants reacted completely, approximately 2 hours. After the reaction, the mixture was concentrated under reduced pressure at room temperature to remove acetonitrile and diethylamine, with DCM added repeatedly for further concentration under reduced pressure to remove diethylamine to the greatest extent possible. After the solvent was evaporated to dryness, 6 mL of ethyl acetate was added to dissolve the product. Then, petroleum ether was added while shaking, resulting in the appearance of a white solid. Petroleum ether was added dropwise until no target product was found in the supernatant. A total of 48 mL of petroleum ether (V) was added. PE :V EA =8:1), centrifuge to separate the solid and liquid phases, wash until there are no impurities in the supernatant, and obtain the deprotected product, a white foamy solid.

[0051] Structural characterization data of Fmoc-His(Trt)-Lys(Boc)-DMDPM: 1 H NMR (400 MHz, DMSO- d 6) δ8.42 (d, J = 7.5 Hz, 1H), 7.93 – 7.82 (m, 6H), 7.73 – 7.56 (m, 6H), 7.52 (s,4H), 7.30 (d, J = 7.1 Hz, 10H), 7.20 (s, 8H), 7.02 (d, J = 8.3 Hz, 6H), 6.76 (d, J= 40.0 Hz, 2H), 6.50 (d, J = 18.1 Hz, 2H), 4.34 (s, 2H), 4.20 – 4.06 (m, 3H), 3.71 (s, 6H), 2.88 – 2.63 (m, 4H), 1.68 (s, 1H), 1.55 (s, 1H), 1.34 (s, 13H). 13 C NMR (126 MHz, DMSO- d 6) δ 172.02, 170.49, 161.19, 157.93, 155.75, 150.53,144.61, 142.76, 141.13, 139.46, 138.66, 133.77, 131.95, 130.74, 129.71,129.45, 129.34, 128.51, 128.35, 128.09, 127.76, 127.52, 125.75, 121.85,120.79, 120.50, 110.21, 105.19, 99.02, 77.79, 74.47, 69.86, 66.65, 60.88, 59.16, 56.07, 55.65, 55.39, 47.11, 31.18, 29.10, 27.52, 22.09. 31 P NMR (202MHz, DMSO- d 6) δ 29.30. Structural characterization data of H-His(Trt)-Lys(Boc)-DMDPM: 1 H NMR (400 MHz, DMSO- d 6) δ8.29 (s, 1H), 7.89 (s, 4H), 7.61 (s, 2H), 7.54 (s, 6H), 7.36 (s, 12H), 7.21 (s, 4H), 7.08 (s, 6H), 6.86 (d, J = 13.3 Hz, 1H), 6.69 (s, 1H), 6.52 (d, J = 16.9Hz, 2H), 4.31 (s, 1H), 3.72 (s, 6H), 3.49 (s, 1H), 2.81 (s, 4H), 1.63 (d, J =38.5 Hz, 2H), 1.36 (s, 13H). 13C NMR (126 MHz, DMSO- d 6) δ 173.57, 171.22,160.46, 157.91, 156.03, 149.97, 143.60, 138.20, 138.15, 133.68, 131.96,131.88, 130.34, 129.71, 129.44, 129.34, 128.61, 128.48, 128.40, 120.83,119.90, 103.98, 98.55, 78.39, 74.47, 71.30, 56.09, 55.67, 55.17, 52.15,40.26, 33.98, 31.63, 29.99, 28.73, 23.98. 31 P NMR (162 MHz, DMSO- d 6) δ 29.33. Experimental synthesis steps of H-His(Trt)-His(Trt)-Lys(Boc)-DMDPM: Accurately weigh Fmoc-His(Trt)-OH (1.80 g, 2.90 mmol, 1.1 eq), EDCI (554 mg, 2.90 mmol, 1.1 eq), HOBt (392 mg, 2.90 mmol, 1.1 eq), and DIEA (516 µL, 2.90 mmol, 1.1 eq), dissolve in 25 mL dichloromethane, stir at 0 °C for 0.5 h to activate the amino acids, then add the compound (3.4 g, 2.64 mmol, 1 eq). Monitor the reaction progress by TLC (evolving solvent DCM:MeOH = 20:1). After approximately 3 h of reaction, the starting material is completely reacted. Stop the reaction, evaporate the solvent, and directly proceed to the next step. Dissolve the crude product in 18 mL acetonitrile, add 6 mL diethylamine (V... MeCN / V DEA =3:1), TCL monitoring throughout (developing solvent DCM:MeOH=10:1), until the reactants reacted completely, approximately 2 hours. After the reaction, the mixture was concentrated under reduced pressure at room temperature to remove acetonitrile and diethylamine, with dichloromethane added multiple times for further concentration under reduced pressure to remove diethylamine to the greatest extent. 50 mL of dichloromethane was added to dissolve the mixture, and it was washed three times with 40 mL of saturated sodium bicarbonate solution and saturated brine, respectively. The organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated. After the solvent was evaporated to dryness, 6 mL of ethyl acetate was added to dissolve the mixture. Then, petroleum ether was added while shaking, resulting in the appearance of a white solid. Petroleum ether was added dropwise until no target product was found in the supernatant. A total of 48 mL of petroleum ether (V) was added. PE :V EA=8:1), centrifuge to separate the solid and liquid phases, wash until there are no impurities in the supernatant, and obtain the deprotected product, which is white and foamy.

[0052] Structural characterization data of H-His(Trt)-His(Trt)-Lys(Boc)-DMDPM: 1 H NMR (500 MHz, DMSO- d 6) δ 8.50 (d, J = 7.8 Hz, 1H), 8.09 (d, J = 7.6 Hz, 1H), 7.92 – 7.82 (m,4H), 7.59 (t, J = 6.6 Hz, 2H), 7.57 – 7.47 (m, 4H), 7.45 – 7.30 (m, 18H), 7.17(s, 4H), 7.07 (dd, J = 7.8, 2.0 Hz, 7H), 7.02 (dd, J = 6.9, 1.6 Hz, 7H), 6.77 (s,1H), 6.62 (d, J = 5.3 Hz, 2H), 6.51 (dd, J = 5.0, 2.4 Hz, 1H), 6.46 (dd, J = 8.5, 2.3 Hz, 1H), 4.54 (t, J = 6.6 Hz, 1H), 4.26 (d, J = 5.6 Hz, 1H), 3.70 (d, J = 3.2Hz, 6H), 2.80 (d, J = 3.8 Hz, 3H), 2.67 (d, J = 9.0 Hz, 2H), 1.76 – 1.60 (m, 1H), 1.53 (dt, J = 14.5, 6.5 Hz, 1H), 1.34 (s, 13H). 13C NMR (126 MHz, DMSO- d6) δ173.95, 171.92, 171.09, 160.72, 157.65, 155.50, 149.51, 142.27, 138.33,137.79, 136.88, 133.69, 131.95, 131.87, 129.68, 129.45, 129.34, 128.61,128.52, 128.40, 128.33, 121.42, 120.78, 119.44, 119.34, 105.84, 98.32, 76.40,74.84, 71.33, 56.05, 55.65, 55.34, 51.15, 40.26, 33.53, 32.34, 31.26, 28.73, 23.51. 31 P NMR (202 MHz, DMSO- d 6) δ 29.25. Subsequent peptide synthesis protocols repeat the above steps, with amino acids, EDCI, HOBt, and DIEA being coupled to four Fmoc-His(Trt)-OH groups in a 1.1 equivalence excess ratio.

[0053] This section utilizes the DMDPM tag to obtain the HHHHHHK-DMDPM structure in high yield, achieving the synthesis of H6K short peptides and verifying the feasibility and stability of the DMDPM tag in peptide synthesis.

[0054] The structural characterization data of each intermediate are as follows: Characterization data for H-His(Trt)-His(Trt)-His(Trt)-Lys(Boc)-DMDPM: 1 H NMR (500MHz, DMSO- d 6) δ 8.45 (t, J = 8.6 Hz, 1H), 8.22 (s, 1H), 7.86 (dd, J = 11.0, 7.2Hz, 4H), 7.58 (d, J = 5.2 Hz, 2H), 7.55 – 7.46 (m, 4H), 7.45 – 7.11 (m, 37H), 7.14 – 6.86 (m, 20H), 6.79 (s, 1H), 6.59 (d, J= 21.1 Hz, 2H), 4.57 (s, 2H), 4.32 (s, 1H), 4.20 (s, 1H), 3.77 – 3.62 (m, 6H), 2.78 (d, J = 9.8 Hz, 4H), 2.70(s, 2H), 1.64 (s, 1H), 1.50 (s, 1H), 1.32 (s, 13H). 31 P NMR (202 MHz, DMSO- d 6)δ 29.21. Structural characterization data of H-His(Trt)-His(Trt)-His(Trt)-His(Trt)-Lys(Boc)-DMDPM: 1 H NMR (500 MHz, DMSO- d 6) δ 8.15 (s, 1H), 8.08 (d, J = 17.9 Hz, 1H), 7.92 – 7.81(m, 4H), 7.64 – 7.50 (m, 2H), 7.49 (s, 4H), 7.45 – 7.12 (m, 45H), 7.02 (dd, J =24.0, 6.6 Hz, 25H), 6.82 (d, J = 2.7 Hz, 1H), 6.57 (d, J = 17.2 Hz, 2H), 4.49 (s,1H), 4.35 (d, J = 28.1 Hz, 2H), 4.12 (s, 1H), 3.68 (d, J = 11.6 Hz, 6H), 2.82 –2.66 (m, 10H), 1.55 (d, J = 36.8 Hz, 2H), 1.29 (s, 13H). 31 P NMR (202 MHz, DMSO- d 6) δ 29.19. Structural characterization data of H-His(Trt)-His(Trt)-His(Trt)-His(Trt)-Lys(Boc)-DMDPM: 1 H NMR (500 MHz, DMSO- d6) δ 8.46 (s, 1H), 8.15 (s, 1H), 8.01 (s, 1H), 7.85 (dd, J = 12.0, 7.9 Hz, 4H), 7.56 (d, J = 6.3 Hz, 2H), 7.55 – 7.45 (m, 4H), 7.34 – 7.08 (m, 58H), 6.99 (t, J = 7.9 Hz, 33H), 6.81 (s, 1H), 6.56 (d, J = 19.5 Hz, 2H), 4.54 (s, 1H), 4.26 (t, J = 51.6 Hz, 3H), 3.69 (d, J = 11.3 Hz, 6H), 2.79 (s, 10H), 2.68 – 2.54 (m, 2H), 1.56 (d, J = 43.2 Hz, 2H), 1.28 (s, 13H). 31 P NMR (202 MHz, DMSO - d 6) δ 29.20. Structural characterization data of H - His(Trt)-His(Trt)-His(Trt)-His(Trt)-His(Trt)-His(Trt)-Lys(Boc)-DMDPM: 1 H NMR (500 MHz, DMSO - d 6) δ 8.17 (s, 1H), 8.04 (s, 1H), 7.85 (dd, J = 12.2, 7.8 Hz, 4H), 7.56 (d, J = 3.1 Hz, 2H), 7.51 – 7.45 (m, 4H), 7.22 (t, J = 9.8 Hz, 57H), 7.04 – 6.93 (m, 45H), 6.81 (s, 1H), 6.55 (d, J = 5.5 Hz, 2H), 4.57 – 4.42 (m, 1H), 4.24 (s, 4H), 3.68 (d, J = 12.1 Hz, 6H), 2.89 – 2.58 (m, 14H), 1.56 (d, J= 43.5 Hz, 2H), 1.29 (s, 13H). 13 C NMR (126 MHz, DMSO- d 6) δ 171.28, 170.07, 169.23, 155.71, 154.11, 152.81, 142.73, 139.67, 138.38,133.72, 131.93, 131.85, 131.00, 129.66, 129.40, 129.30, 128.56, 128.48,121.32, 119.12, 112.82, 101.32, 78.70, 76.69, 74.87, 60.23, 56.43, 55.60,53.34, 40.27, 30.31, 28.14, 22.99, 20.88, 13.93. 31 P NMR (202 MHz, DMSO- d 6) δ29.17. Synthetic protocol for peptide chain cleavage from the tag: Weigh compound H-His(Trt)-His(Trt)-His(Trt)-His(Trt)-His(Trt)-Lys(Boc)-DMDPM (500 mg, 0.17 mmol) and add it to a 10 mL TFA / TIS / H2O (v / v / v, 95:2.5:2.5) mixture. Stir at room temperature and monitor the reaction progress by TLC. After 3 h, the starting material has completely reacted. Add dichloromethane to the mixture and remove TFA, H2O, and TIS by rotary evaporation at 40 °C. Repeat the above operation by adding dichloromethane several times. After rotary drying, add cold diethyl ether and sonicate. A pale yellow solid appears. Crush the precipitate by ultrasonic vibration and centrifuge to obtain the cleaved crude peptide. Continue to add cold diethyl ether and wash with ultrasonication for about three times or more until there are no impurities in the supernatant. Centrifuge to obtain the cleaved peptide as a pale yellow solid. The ether phase was concentrated and purified by column chromatography to obtain the cleaved DMDPM tag.

[0055] H6K structural characterization data: 1 H NMR (500 MHz, DMSO- d 6) δ 14.46 (s, 7H), 8.96 (d, J = 12.4 Hz, 6H), 8.71 (d, J = 7.8 Hz, 1H), 8.56 (s, 2H), 8.51 (d, J = 7.6 Hz, 1H), 8.43 (d,J = 7.3 Hz, 1H), 7.88 (s, 3H), 7.37 (d, J = 11.7 Hz, 2H), 7.31 (d, J = 5.2Hz, 4H), 4.64 (td, J = 12.8, 5.8 Hz, 5H), 4.25 – 4.13 (m, 2H), 3.25 – 3.05 (m,7H), 3.05 – 2.89 (m, 5H), 2.77 (s, 2H), 1.68 – 1.46 (m, 4H), 1.39 – 1.33 (m,2H). HRMS (ESI) m / z calcd for C 42 H 56 N 20 O8Na + (M+Na) + 991.4482198, found 991.44861. See Figure 3 The HPLC analysis results for H6K are shown in the figure.

[0056] Example 2 Similar to Example 1, except that the last amino acid in Example 1 was replaced with Boc-His(Trt)-OH, and the coupling product was treated directly with TFA / TIS / H2O (v / v / v, 95:2.5:2.5) reagent without de-Boc removal. Other steps were performed as in Example 1 for synthesis and preparation, followed by separation and purification to obtain the H6K product.

[0057] Example 3 Similar to Example 1, except that the final treatment reagent TFA / TIS / H2O (v / v / v, 95:2.5:2.5) from Example 1 was treated with a 95% TFA solution in dichloromethane. Other steps were performed as in Example 1 or 2, followed by synthesis and purification to obtain the H6K product.

[0058] It should be noted that, from the perspective of the molecular tag's structural design, the presence of a diphenoxyphosphonoyl group at the para position of one benzene ring imparts to DMDPM the property of assisting in the precipitation and separation of peptides. Meanwhile, the presence of two electron-donating methoxy groups at the ortho and para positions of the other benzene ring enhances the esterification activity of the diphenylmethanol hydroxyl group through the electron-donating effect and also regulates the molecular tag's adaptability to organic solvents. The combined effect of these two methoxy groups is to enhance the esterification activity of the benzyl alcohol hydroxyl group during coupling with amino acids, and to facilitate the cleavage activity of the peptide chain by trifluoroacetic acid after peptide chain elongation. Furthermore, the cleaved molecular tag fragments can be recycled and reused. On the other hand, the availability of raw materials and the simplicity of the process for synthesizing DMDPM are also considered. In summary, the DMDPM provided by this invention has significant advantages in terms of molecular structure design and process optimization.

[0059] The above embodiments are merely some examples listed to facilitate understanding of the synthesis and application methods of the materials of the present invention, and are not intended to limit the present invention. It is understood that those skilled in the art can easily make appropriate modifications to this structure; therefore, any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A molecularly labeled DMDPM compound, characterized in that, The structural formula is as follows: Among them, the DMDPM compound is 2,4-dimethoxy-4'-diphenylphosphonodiphenylethanol.

2. A method for preparing the molecularly labeled DMDPM compound according to claim 1, characterized in that, Includes the following steps: 2,4-Dimethoxy-4'-hydroxybenzophenone was dissolved in tetrahydrofuran, sealed, placed in an ice bath, and then triethylamine was added. After mixing thoroughly, diphenylphosphine chloride diluted with tetrahydrofuran was added. After the reaction was completed, a white solid product was obtained. The white solid product was dissolved in a mixed solution of methanol and tetrahydrofuran. The mixed solution was placed in an ice bath, and NaBH4 was added in several portions. The mixture was then sealed and stirred under ice bath conditions. After the reaction was completed, the mixture was quenched with saturated NH4Cl solution. The precipitate was removed by filtration. The filtrate was concentrated under reduced pressure and washed to obtain the DMDPM compound.

3. The use of the molecularly labeled DMDPM compound of claim 1 in the assisted homogeneous synthesis of heptapeptide H6K.

4. A method for homogeneous synthesis of heptapeptide H6K assisted by a molecularly labeled DMDPM compound, characterized in that, Includes the following steps: Using DMDPM compound as an auxiliary group, it is reacted with amino acid under the action of a dehydrating coupling agent, so that the C-terminus of the amino acid is connected to the auxiliary group to generate compound A; wherein the amino acid is L-lysine Fmoc-Lys(Boc)-OH with both amino groups protected. The N-terminal Fmoc of compound A was removed to obtain deprotected product B; Fmoc-His(Trt)-OH, EDCI, HOBt, and DIEA were dissolved in dichloromethane, and then deprotected product B was added to carry out a coupling reaction to obtain compound C. Fmoc was removed from compound C to obtain deprotected product D; Fmoc-His(Trt)-OH, EDCI, HOBt, and DIEA were dissolved in dichloromethane, and then deprotected product D was added for coupling reaction. After the reaction was completed, Fmoc was removed to obtain deprotected product E. Subsequently, this step was repeated, and deprotected product D was replaced by deprotected product E to continue coupling four Fmoc-His(Trt)-OH to obtain the compound H-His(Trt)-His(Trt)-His(Trt)-His(Trt)-His(Trt)-Lys(Boc)-DMDPM. The molecular tag DMDPM on the compound H-His(Trt)-His(Trt)-His(Trt)-His(Trt)-His(Trt)-His(Trt)-Lys(Boc)-DMDPM was cleaved to obtain the heptapeptide H6K.

5. The method for homogeneous synthesis of heptapeptide H6K assisted by the molecularly labeled DMDPM compound according to claim 4, characterized in that, The Fmoc expulsion process includes: The Fmoc-protected compound was dissolved in acetonitrile, and then diethylamine was added to react. After the reaction was completed, the acetonitrile and diethylamine were removed by vacuum concentration at room temperature. DCM was added repeatedly for vacuum concentration to remove diethylamine to the greatest extent. After the solvent was evaporated to dryness, ethyl acetate was added to dissolve the compound. Petroleum ether was then added dropwise while shaking until no target product was found in the supernatant. The solid and liquid phases were separated by centrifugation and washed until no impurities were found in the supernatant to obtain the deprotected product.

6. The method for homogeneous synthesis of heptapeptide H6K assisted by the molecularly labeled DMDPM compound according to claim 4, characterized in that, The dehydrating coupling agent is a 1:1 molar ratio of a dehydrating coupling activator and an alkaline substance; wherein the dehydrating coupling activator is EDCI, and the alkaline substance is DIEA or DMAP. The molar ratio of the amino acid to the auxiliary group is 1 to 1.

1.

7. The method for homogeneous synthesis of heptapeptide H6K assisted by the molecularly labeled DMDPM compound according to claim 4, characterized in that, The molecular structure of the deprotected product B is as follows: The molecular structure of the deprotected product D is as follows:

8. The method for homogeneous synthesis of heptapeptide H6K assisted by the molecularly labeled DMDPM compound according to claim 4, characterized in that, The molecular structural formula of the H-His(Trt)-His(Trt)-His(Trt)-His(Trt)-His(Trt)-His(Trt)-Lys(Boc)-DMDPM compound is as follows:

9. The method for homogeneous synthesis of heptapeptide H6K assisted by the molecularly labeled DMDPM compound according to claim 4, characterized in that, The shearing process includes: The compound H-His(Trt)-His(Trt)-His(Trt)-His(Trt)-His(Trt)-Lys(Boc)-DMDPM was added to a TFA / TIS / H2O mixture and stirred at room temperature. After the reaction was complete, dichloromethane was added to the resulting mixture, and TFA, H2O, and TIS were removed by rotary evaporation at 40°C. This process was repeated with multiple additions of dichloromethane. After rotary drying, cold diethyl ether was added, and the mixture was sonicated. The precipitate was then pulverized by ultrasonic vibration and centrifuged to obtain the cleaved crude peptide. Cold diethyl ether was added again, and the mixture was washed with ultrasonic waves approximately three times or more until the supernatant was free of impurities. After centrifugation, the cleaved heptapeptide H6K was obtained. The ether phase was then concentrated and purified by column chromatography to obtain the cleaved DMDPM tag. The volume ratio of TFA / TIS / H2O was 95:2.5:2.

5.

10. A heptapeptide H6K prepared by the method according to any one of claims 4 to 9, characterized in that, The structural formula of the heptapeptide H6K is as follows:

Images