Liquid phase synthesis method of silk fibroin peptide

Abstract

The application provides a liquid-phase synthesis method of silk fibroin peptide and belongs to the technical field of organic materials. The silk fibroin peptide is shown as formula (I). The application adopts a GA+GA convergent condensation strategy, uses N-Boc-glycine and alanine benzyl ester hydrochloride as starting materials, sequentially constructs dipeptide, tetrapeptide through condensation, deprotection and recondensation, and condenses the prepared GS dipeptide to obtain a hexapeptide skeleton, and finally removes the protecting group to obtain the target product. The application determines the optimal process parameters by screening the connection sequence and condensing agent system, and the purity and yield of each intermediate and final product are high. The application is simple in operation, does not need chromatography purification, is good in batch stability, is easy to scale up, and provides an economic and reliable technical scheme for efficient preparation of silk fibroin characteristic peptide segments. Formula (I).

Description

Technical Field

[0001] This invention belongs to the field of organic materials technology, specifically relating to a liquid-phase synthesis method for silk fibroin peptides. Background Technology

[0002] Silk fibroin is the core protein component of natural silk fibers, accounting for 70-80% of the total weight of silk. As a natural fibrous protein, silk fibroin is composed of 18 amino acids, with glycine, alanine, serine, and tyrosine accounting for over 90%, and glycine being the most abundant at approximately 44%. Studies have shown that the amino acid composition of silk fibroin is as similar as 87% to that of human skin, a characteristic that gives it unique advantages in biomedical and daily chemical applications. From a molecular structure perspective, the heavy chain of silk fibroin contains highly repetitive amino acid sequences, and its β-sheet crystal structure is mainly composed of the six-amino acid repeating sequence GAGAGS (glycine-alanine-glycine-alanine-glycine-serine). In addition, the heavy chain of silk fibroin also contains 11 irregular sequences, each containing approximately 31 amino acid residues. These irregular sequences can form ring structures, altering the orientation of the peptide backbone and promoting the formation of β-sheets between structural units in the crystalline region. Due to its excellent biocompatibility, mechanical properties, and biodegradability, silk fibroin and its related product, silk fibroin peptides, have shown broad application prospects in multiple fields. In the biomedical field, silk fibroin can be used to prepare medical devices such as tissue engineering scaffolds, drug delivery carriers, artificial skin, and artificial corneas. In the daily chemical industry, silk fibroin peptides, as cosmetic additives, have good hygroscopic and moisturizing properties. In addition, silk fibroin peptides also have antibacterial activity, inhibiting the growth of bacteria in the air. In emerging technology fields, silk fibroin is also applied in high-end applications such as biological storage media, flexible electronic devices, and composite reinforcing materials.

[0003] The preparation methods for silk fibroin and silk fibroin peptides are mainly divided into two categories: one is the "extraction method," which extracts regenerated silk fibroin from natural silkworm cocoons; the other is the "synthetic biology method," which uses genetic engineering for recombination expression. The preparation method for silk fibroin peptides primarily uses hydrolysis.

[0004] Natural extraction is currently the most mainstream method in industrial production and laboratory research. Its basic process includes degumming, dissolution, dialysis purification and reshaping. This traditional preparation method has many technical defects that are difficult to overcome: (1) Long preparation cycle and low efficiency: The traditional dialysis desalting process usually takes 2-5 days. Although the repeated freeze-thaw method can reduce costs, the preparation cycle is long. The overall process is time-consuming and labor-intensive, which is difficult to meet the efficiency requirements of large-scale industrial production. (2) Severe degradation of silk fibroin molecules: During the degumming and dissolution process, silk fibroin molecules will be degraded to a certain extent. Although conventional Na2CO3 degumming method can completely remove sericin, it causes significant damage to fibroin, resulting in severe surface corrosion and a sharp drop in the molecular weight of the regenerated fibroin. The decrease in molecular weight directly affects the mechanical properties and application value of the regenerated fibroin material. (3) Low solution concentration and difficulty in subsequent processing: While the conventional dialysis method removes salt ions, a large number of water molecules seep into the dialysis bag, resulting in a generally low concentration of the final fibroin solution. (4) Poor batch stability and difficulty in quality control: Naturally derived fibroin is limited by the periodicity, regionality, and environmental dependence of silkworm breeding. It exhibits significant natural variability in molecular weight, amino acid sequence, higher-order structure, and batch purity and performance consistency, making it difficult to achieve precise control.

[0005] To overcome the limitations of natural extraction methods, researchers have recently attempted to utilize genetic engineering techniques to achieve recombinant expression of silk fibroin through expression systems such as Pichia pastoris. Theoretically, this method can achieve precise synthesis of silk fibroin, yielding products with defined sequences and uniform molecular weights. However, current recombinant expression methods still face the following problems: the molecular weight of recombinant silk fibroin is too small, mostly only one-third or even less of the molecular weight of natural silk fibroin; the gene vector rejects foreign silk fibroin genes, leading to denaturation of the recombinant silk fibroin; and the cost of genetically engineered proteins is too high, even failing to meet the needs of laboratory use.

[0006] The preparation methods for silk fibroin peptides mainly include salting out, acid hydrolysis, alkaline hydrolysis, and enzymatic hydrolysis. Each method has its advantages and disadvantages: salt solution degradation can yield silk fibroin with a molecular weight of tens of thousands, but since it is still a large protein molecule, it affects its absorption and nutritional value; acid hydrolysis can fully hydrolyze silk fibroin, but it will lead to the complete destruction of tryptophan and partial destruction of serine, tyrosine, and threonine; alkaline hydrolysis causes racemization, reducing nutritional value; enzymatic hydrolysis has mild reaction conditions and causes less damage to amino acids, and is currently recognized as the most promising degradation method. However, the hydrolysis efficiency of plant proteases, animal proteases, and microbial proteases used in the currently reported enzymatic hydrolysis is generally not high, and the enzymatic hydrolysis process is complex to control, with precise control of the degree of hydrolysis remaining a technical challenge.

[0007] It is noteworthy that the core unit constituting the β-sheet crystal structure of silk fibroin heavy chains is the GAGAGS hexapeptide repeat sequence, which is a key structural domain endowing silk fibroin with excellent mechanical properties and biological activity. However, the aforementioned natural extraction methods are insufficient to obtain specific oligopeptides with uniform sequences, recombinant expression methods are inefficient due to the rejection of short repeat units by gene vectors, and traditional hydrolysis methods cannot selectively prepare such short peptides with single sequences. Therefore, developing a method for the efficient and controllable synthesis of pure H-GAGAGS-OH is of significant scientific and practical value for elucidating the structure-function relationship of silk fibroin at the molecular level and developing high-purity peptide products based on the smallest functional unit.

[0008] In summary, existing methods for preparing silk fibroin and silk peptides generally suffer from problems such as complex processes, long cycles, severe molecular weight degradation, poor batch stability, and high production costs. These limitations make it difficult to meet the urgent needs of biopharmaceutical, high-end daily chemical, and other application fields for high-purity, high-activity, controllable molecular weight, and batch-stable silk fibroin products. Therefore, developing a method for preparing silk fibroin or silk peptides that is simple to operate, has a short cycle time, is environmentally friendly, low-cost, easy to scale up, and can achieve high purity, controllable molecular weight, and batch stability is of great significance for expanding their applications in cutting-edge fields such as biopharmaceuticals and daily chemical products. Summary of the Invention

[0009] To address the problems of low concentration, low yield, uncontrollable molecular weight, and difficulty in large-scale production of existing silk fibroin peptides, the present invention aims to obtain a more economical and efficient liquid-phase synthesis route with high purity, controllable molecular weight, and batch stability, so as to achieve large-scale preparation of silk fibroin peptides.

[0010] The specific technical solution of the present invention is as follows: This invention provides a method for synthesizing silk fibroin peptides, wherein the silk fibroin peptides have the structure shown in formula (I), and the synthesis method is a liquid-phase synthesis method, comprising the following steps: Formula (I) (a) R 1 -GAGA-OR 2 Remove R 2 Protecting base, to obtain R 1 -GAGA-OH; (b) With amino protecting group as R 3 The glycine and carboxyl protecting groups are R 4 Using serine salt as a raw material, condensation is carried out in the presence of a condensing agent and an organic base to obtain R. 3 -GS-OR 4 ; (c) Take the R obtained in step (b) 3 -GS-OR 4 Remove R 3 Protecting group, yielding H-GS-OR 4 ; (d) Take the R obtained in step (a) 1 -GAGA-OH and H-GS-OR obtained in step (c) 4 Condensation in the presence of a condensing agent and an organic base yields R. 1 -GAGAGS-OR 4 ; (e) Take the R obtained in step (d) 1 -GAGAGS-OR 4 Remove R 4 Protecting base, to obtain R 1 -GAGAGS-OH; (f) Take the R obtained in step (e) 1 -GAGAGS-OH removal R 1 Protecting group, yielding silk fibroin peptide as shown in Formula I; Wherein, the R 1 R 3 R is an amino protecting group. 2 R 4 It is a carboxyl protecting group.

[0011] Furthermore, the R 1 R 3 Each is independently selected from any one of tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), or methoxycarbonyl (Fmoc); the R 2 R 4 Each is independently selected from any one of benzyl (Bn), methyl, ethyl or tert-butyl.

[0012] Furthermore, the R 1 R 3 It is tert-butyloxycarbonyl (Boc); the R 2 R 4 It is benzyl (Bn).

[0013] Furthermore, the alanine salt is alanine hydrochloride; the serine salt is serine hydrochloride.

[0014] Further, in step (a), the R 1 -GAGA-OR 2 The preparation method includes the following steps: (1) With amino protecting group as R 1 The glycine and carboxyl protecting groups are R 2Using alanine salt as a raw material, condensation is carried out in the presence of a condensing agent and an organic base to obtain R. 1 -GA-OR 2 ; (2) Take the R obtained in step (1) 1 -GA-OR 2 Remove R 2 Protecting base, to obtain R 1 -GA-OH; (3) Take the R obtained in step (1) 1 -GA-OR 2 Remove R 1 Protecting group, yielding H-GA-OR 2 ; (4) Take the R obtained in step (2) 1 -GA-OH reacts with an activating agent and a condensing agent to give an activated intermediate; (5) Combine the activated intermediate obtained in step (4) with the H-GA-OR obtained in step (3). 2 Condensation in the presence of an organic base yields R 1 -GAGA-OR 2 .

[0015] Further, the condensing agent mentioned in steps (1), (d), and (d) is selected from any one or a combination of 1-hydroxybenzotriazole (HOBT), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU); the organic base is selected from any one of triethylamine, N,N-diisopropylethylamine (DIEA), or N-methylmorpholine. The removal of R described in steps (2), (a), and (e) 2 Or R 4 Each protecting group is independently prepared using catalytic hydrogenolysis. The removal of R described in steps (3), (c), and (f) 1 Or R 3 Each protecting group is independently prepared using an acidic reagent; The activating agent in step (4) is selected from any one of pentafluorophenol (PFP), N-hydroxysuccinimide, N-(benzyloxycarbonyloxy)succinimide, 9-fluorenylmethoxycarbonylsuccinimide, ethyl 2-oxime cyanoacetate, O-benzotriazole-tetramethylurea hexafluorophosphate, and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate; the condensing agent is selected from any one or a combination of 1-hydroxybenzotriazole, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate. The organic base mentioned in step (5) is selected from any one of triethylamine, N,N-diisopropylethylamine or N-methylmorpholine.

[0016] Further, in steps (1) and (b), the condensing agent is a combination of 1-hydroxybenzotriazole and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; the organic base is N,N-diisopropylethylamine; in step (d), the condensing agent is benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, and the organic base is N,N-diisopropylethylamine; In steps (2), (a), and (e), the catalyst in the catalytic hydrogenolysis method is carbon-supported palladium, and the hydrogen source is hydrogen gas. In step (3), the acidic reagent is trifluoroacetic acid; in steps (c) and (f), the acidic reagent is 1,4-dioxane hydrochloride. In step (4), the activating agent is pentafluorophenol; the condensing agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; In step (5), the organic base is N,N-diisopropylethylamine.

[0017] Furthermore, each step of the reaction uses an organic solvent independently; In step (1), the organic solvent and the amino protecting group are R. 1 The molar ratio of glycine, alanine salt, 1-hydroxybenzotriazole, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N,N-diisopropylethylamine is (8~12):1:(1.1~1.3):(1.0~1.2):(1.3~1.7):(1.0~1.4); the reaction temperature is 0~10℃ and the time is 1~3h. In step (3), the organic solvent, R 1 -GA-OR 2 The molar ratio of trifluoroacetic acid to trifluoroacetic acid is (8~12):1:(3~10); the reaction time is 1~3 hours. In step (4), the organic solvent, R 1 The molar ratio of GA-OH, pentafluorophenol, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is (8~12): 1: (1.0~1.2): (1.1~1.3); the reaction time is 1~3 h; In step (5), the organic solvent, the activation intermediate, and H-GA-OR 2 The molar ratio of N,N-diisopropylethylamine is (8~12):1:(0.9~1.1):(2~8); the reaction time is 1~3 h; In step (b), the organic solvent and the amino protecting group are R. 3 The molar ratio of glycine, serine, 1-hydroxybenzotriazole, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N,N-diisopropylethylamine is (8~12):1:(1.1~1.3):(1.0~1.2):(1.1~1.3):(1.3~1.7); the reaction temperature is 0~10℃ and the time is 1~3h. In step (c), the organic solvent, R 3 -GS-OR 4 The molar ratio of 1,4-dioxane hydrochloride to 1,4-dioxane is (8-12):1:(3-10); the reaction time is 1-3 hours. In step (d), the organic solvent, R 1 -GAGA-OH、H-GS-OR 4 The molar ratio of benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine is (8~12):1:(1.0~1.2):(1.1~1.5):(2~8); the reaction time is 1~3h.

[0018] Furthermore, steps (2), (a), and (e) are all carried out under organic solvent conditions, where the organic solvent and R... a -peptide-OR b The molar ratio of palladium to carbon-supported palladium is (8~12):1:(0.005~0.02); the reaction time is 3~8 h; the R... a -peptide-OR b For R 1 -GA-OR 2 R 1 -GAGA-OR 2 Or R 1 -GAGAGS-OR 4 .

[0019] Furthermore, step (f) is carried out under organic solvent conditions, wherein the organic solvent, R... 1 The molar ratio of -GAGAGS-OH to 1,4-dioxane hydrochloride is (8~12):1:(3~10); the reaction time is 1~3h.

[0020] Furthermore, in addition to the deprotection reaction, after each reaction step is completed, one or more of the following purification steps are also included: extraction, drying with anhydrous sodium sulfate, concentration under reduced pressure, filtration, precipitation, and recrystallization.

[0021] Furthermore, in step (5), R is obtained. 1-GAGA-OR 2 The process also includes a post-processing step, wherein the post-processing adopts the methyl tert-butyl ether precipitation method; In step (d), R is obtained. 1 -GAGAGS-OR 4 The process also includes a post-processing step, which employs recrystallization using a mixed solvent of methanol and ethyl acetate.

[0022] The present invention has achieved the following beneficial effects: (1) High product purity and stable yield: The purity of each intermediate and final product of this invention is above 95% (some steps ≥ 98%). The yields of key steps are Boc-GA-OBn > 92%, Boc-GAGA-OBn > 85%, and Boc-GAGAGS-OBn > 80%, respectively. The deprotection steps are all > 95%, and the batch reproducibility is good, overcoming the defect of poor batch stability of natural extraction method.

[0023] (2) Precise and controllable molecular weight: Directly synthesize GAGAGS characteristic peptides with defined sequences, resulting in uniform molecular weight and clear structure, avoiding the problems of wide molecular weight distribution and poor product uniformity caused by natural hydrolysis or recombinant expression methods.

[0024] (3) The process is highly scalable: the reaction conditions are mild, and the operation units are all classic condensation, deprotection, extraction, precipitation or recrystallization. No chromatographic purification or special equipment is required, making it easy to industrialize.

[0025] (4) Cost controllable: The raw materials are all commercially available reagents, and the production cost is low.

[0026] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention.

[0027] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0028] Figure 1 The HPLC chromatogram of Boc-GA-OBn is shown.

[0029] Figure 2 The HPLC chromatograms are shown for (a) Boc-GA-OH and (b) Boc-GA-PFP, respectively.

[0030] Figure 3 The HPLC chromatogram of H-GA-OBn is shown.

[0031] Figure 4 The images show the HPLC chromatograms of Boc-GAGA-OBn.

[0032] Figure 5 The HPLC chromatograms are shown for (a) Boc-GS-OBn and (b) H-GS-OBn, respectively.

[0033] Figure 6 The HPLC chromatograms are shown for (a) Boc-GAGAGS-OBn and (b) Boc-GAGAGS-OH, respectively.

[0034] Figure 7 The figures are (a) the HPLC chromatogram of H-GAGAGS-OH and (b) the LC-MS chromatogram of H-GAGAGS-OH.

[0035] Figure 8 The HPLC chromatogram of Boc-GAGA-OBn synthesized using the HOBT system is shown.

[0036] Figure 9 The HPLC chromatogram of Boc-GAGAGS-OBn synthesized using the DMTMM system is shown.

[0037] Figure 10 The HPLC chromatogram of Boc-GAGAGS-OBn synthesized using the HOBT / EDC system is shown.

[0038] Figure 11 The HPLC chromatogram of Boc-GAGAGS-OBn synthesized using the PFP active intermediate is shown. Detailed Implementation

[0039] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0040] Example 1: Synthesis of H-GAGAGS-OH The structure of H-GAGAGS-OH is as follows: (1) Synthesis of Boc-GA-OBn N-Boc-glycine (Boc-G-OH), alanine benzyl ester hydrochloride (HA-OBn·HCl), HOBT, N,N-diisopropylethylamine (DIEA), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) were added sequentially to dichloromethane (DCM) at 0–10 °C. After stirring and dissolving, the mixture was reacted for 2 h. The molar ratio of each reagent to solvent was DCM: Boc-G-OH: HA-OBn·HCl: HOBT: DIEA: EDC·HCl = 10: 1: 1.2: 1.05: 1.5: 1.2. After stirring for 2 h, the mixture was extracted twice with saturated NaHCO3 solution, twice with 0.1 M KHSO4 aqueous solution, and washed once with saturated sodium chloride aqueous solution. The organic phase was collected, dried with anhydrous Na2SO4, and concentrated under reduced pressure to obtain an oily liquid of Boc-GA-OBn with a yield higher than 92% and a purity higher than 97%. The HPLC chromatogram of Boc-GA-OBn is as follows: Figure 1 As shown.

[0041] (2) Synthesis of Boc-GA-OH The Boc-GA-OBn obtained in step (1) was dissolved in methanol (MeOH), and a catalyst supported on carbon palladium (Pd / C) was added, wherein the equivalent ratio of Boc-GA-OBn:Pd / C = 1:0.01, and the concentration of Boc-GA-OBn in MeOH was 0.1 g / ml. Hydrogen gas was then introduced, and the reaction was carried out for 5 hours. After the reaction was completed, the mixture was filtered under reduced pressure, the solvent was evaporated, and then vacuum dried to obtain solid Boc-GA-OH. The yield was higher than 95%, and the purity was higher than 98%. The HPLC chromatogram of Boc-GA-OH is shown below. Figure 2 As shown in a.

[0042] (3) Synthesis of H-GA-OBn The Boc-GA-OBn obtained in step (1) was dissolved in DCM, and trifluoroacetic acid (TFA) was added. The equivalent ratio of each reagent to solvent was DCM:Boc-GA-OBn:TFA = 10:1:(3~10). The reaction was carried out for 2 hours. After the reaction was completed, the solution was concentrated under reduced pressure to an oily liquid of H-GA-OBn with a yield higher than 95% and a purity higher than 98%. The HPLC chromatogram of H-GA-OBn is shown below. Figure 3 As shown.

[0043] (4) Synthesis of Boc-GA-PFP The Boc-GA-OH obtained in step (2) was dissolved in DCM with pentafluorophenol (PFP) and EDC·HCl, wherein the equivalent ratio of each reagent and solvent was DCM: Boc-GA-OH: PFP: EDC·HCl = 10: 1: 1.05: 1.2. The mixture was stirred for 2 hours. After the reaction was completed, half a volume of water was added to the reaction solution for extraction twice to remove excess EDC·HCl and some water-soluble impurities. The DCM layer was dehydrated using anhydrous sodium sulfate and then filtered to obtain a DCM solution containing Boc-GA-PFP. Subsequently, the DCM solvent was removed by rotary evaporation to obtain Boc-GA-PFP with a yield higher than 90% and a purity higher than 95%. The HPLC chromatogram of Boc-GA-PFP is shown below. Figure 2 As shown in b.

[0044] (5) Synthesis of Boc-GAGA-OBn The Boc-GA-PFP obtained in step (4) was dissolved in DCM, and H-GA-OBn and N,N-diisopropylethylamine (DIEA) obtained in step (3) were added sequentially. The equivalent ratio of each reagent and solvent was DCM: Boc-GA-PFP: H-GA-OBn:DIEA = 10: 1.05: 1: (2~8). The mixture was stirred for 2 hours. After the reaction was completed, the mixture was extracted twice with saturated NaHCO3 solution, twice with 0.1M KHSO4 aqueous solution, and washed once with saturated sodium chloride aqueous solution. The organic phase was collected, dried with anhydrous Na2SO4, and concentrated under reduced pressure to obtain a pale yellow oily liquid. The concentrated filtrate was slowly added dropwise to methyl tert-butyl ether (MTBE), and a white solid precipitated. After filtration and vacuum drying, Boc-GAGA-OBn solid was obtained with a yield higher than 85% and a purity higher than 96%. The HPLC chromatogram of Boc-GAGA-OBn is shown below. Figure 4 As shown.

[0045] (6) Synthesis of Boc-GAGA-OH The Boc-GAGA-OBn obtained in step (5) was dissolved in methanol (MeOH), and a catalyst supported on carbon palladium (Pd / C) was added, wherein the equivalent ratio of Boc-GAGA-OBn:Pd / C = 1:0.01, and the concentration of Boc-GAGA-OBn in MeOH was 0.1 g / ml. Hydrogen gas was purged, and the reaction was carried out for 5 hours. After the reaction was completed, the mixture was filtered under reduced pressure, the solvent was evaporated, and then vacuum dried to obtain solid Boc-GAGA-OH with a yield higher than 95% and a purity higher than 98%.

[0046] (7) Synthesis of Boc-GS-OBn N-Boc-glycine (Boc-G-OH), serine benzyl ester hydrochloride (HS-OBn·HCl), HOBT, N,N-diisopropylethylamine (DIEA), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) were added sequentially to dichloromethane (DCM) at 0-10℃. After stirring and dissolving, the mixture was reacted for 2 hours. The molar ratio of each reagent to solvent was DCM: Boc-G-OH: HS-OBn·HCl: HOBT: DIEA: EDC·HCl = 10: 1: 1.2: 1.05: 1.5: 1.2. After stirring for 2 hours, the mixture was extracted twice with saturated NaHCO3 aqueous solution, twice with 0.1M KHSO4 aqueous solution, and washed once with saturated sodium chloride aqueous solution. The organic phase was collected, dried with anhydrous Na2SO4, and concentrated under reduced pressure to obtain an oily liquid of Boc-GS-OBn with a yield higher than 90% and a purity higher than 97%. The HPLC chromatogram of Boc-GS-OBn is as follows: Figure 5 As shown in a.

[0047] (8) Synthesis of H-GS-OBn The Boc-GS-OBn obtained in step (7) was dissolved in DCM, and 1,4-dioxane hydrochloride solution (4M) was added. The equivalent ratio of each reagent to solvent was DCM: Boc-GS-OBn: 1,4-dioxane hydrochloride solution (4M) = 10: 1: (3~10). The reaction was carried out for 2 hours. After the reaction was completed, H-GS-OBn white solid was obtained by filtration with a yield higher than 95% and a purity higher than 98%. The HPLC chromatogram of H-GS-OBn is shown below. Figure 5 As shown in b.

[0048] (9) Synthesis of Boc-GAGAGS-OBn The Boc-GAGA-OH obtained in step (6) was dissolved in DMF, and HCl·H-GS-OBn, HBTU, and N,N-diisopropylethylamine (DIEA) obtained in step (8) were added sequentially. The equivalent ratio of each reagent and solvent was DMF: Boc-GAGA-OH:HCl·H-GS-OBn: HBTU: DIEA = 10: 1: 1.05: 1.2: (2~8). The mixture was stirred for 2 hours. After the reaction was completed, pure water and DCM were added and extracted three times. The ratio of pure water to DCM was 2:1. The DCM layer was collected and extracted twice with saturated NaHCO3 aqueous solution, twice with 0.1M KHSO4 aqueous solution, and washed once with saturated sodium chloride aqueous solution. The organic phase was collected, dried with anhydrous Na2SO4, and concentrated under reduced pressure to obtain a solid crude product. The product was purified by recrystallization at 60℃ using methanol (MeOH) and ethyl acetate (EA). Upon cooling, a white solid precipitated, which was then filtered and dried to obtain Boc-GAGAGS-OBn solid. The yield was higher than 80%, and the purity was higher than 95%. The HPLC chromatogram of Boc-GAGAGS-OBn is shown below. Figure 6 As shown in a.

[0049] (10) Synthesis of Boc-GAGAGS-OH The Boc-GAGAGS-OBn obtained in step (9) was dissolved in methanol (MeOH), and a catalyst supported on carbon palladium (Pd / C) was added, wherein the equivalent ratio of Boc-GAGAGS-OBn:Pd / C = 1:0.01, the concentration of Boc-GAGAGS-OBn in MeOH was 0.1 g / ml, and hydrogen gas was introduced. The reaction was carried out for 5 hours. After the reaction was completed, the mixture was filtered under reduced pressure, the solvent was evaporated, and then dried under vacuum to obtain solid Boc-GAGAGS-OH. The yield was higher than 95%, and the purity was higher than 98%. The HPLC chromatogram of Boc-GAGAGS-OH is shown below. Figure 6 As shown in b.

[0050] (11) Synthesis of H-GAGAGS-OH The Boc-GAGAGS-OH obtained in step (10) was dissolved in dioxane, and 1,4-dioxane hydrochloric acid solution (4M) was added. The equivalent ratio of each reagent to solvent was dioxane: Boc-GAGAGS-OH: 1,4-dioxane hydrochloric acid solution (4M) = 10: 1: (3~10). The reaction was carried out for 2 hours. After the reaction was completed, H-GAGAGS-OH white solid was obtained by filtration with a yield higher than 95% and a purity higher than 98%. The HPLC chromatogram and LC-MS chromatogram of H-GAGAGS-OH are shown below. Figure 7 As shown in a and b.

[0051] The following experimental examples demonstrate the beneficial effects of the present invention.

[0052] Experiment 1: Screening of amino acid linkage sequence 1. Boc-GAGA-OH is synthesized from Boc-GAGAG-OH by reacting with HCl·HG-OH. Boc-GAGA-OH was prepared according to the steps described in Example 1. Boc-GAGA-OH was then reacted with PFP and EDC to synthesize Boc-GAGA-PFP, where the equivalent ratio of each solvent to reagent was DCM:Boc-GAGA-OH:PFP:EDC = 10:1:1.1:1.2. The reaction was stirred for 2 hours. After the reaction was complete, the mixture was washed twice with water, dried with anhydrous Na2SO4, and concentrated under reduced pressure to obtain a pale yellow oily liquid. Boc-GAGA-PFP was then reacted with HCl·HG-OH to synthesize Boc-GAGAG-OH, where the equivalent ratio of each reagent to solvent was DCM:Boc-GAGA-PFP:HCl·HG-OH:DIEA = 10:1:1.2:1.5. The reaction was stirred for 2 hours, and HPLC analysis of the reaction solution showed no product formation.

[0053] 2. Boc-GAGA-OH reacts with HCl·HG-OBn to synthesize Boc-GAGAG-OBn. Boc-GAGA-OH was prepared according to the steps described in Example 1. Boc-GAGA-OH was then reacted with HCl·HG-OBn to synthesize Boc-GAGAG-OBn, where the equivalent ratio of each reagent and solvent was DCM: Boc-GAGA-OH: HCl·HG-OBn: HOBT: EDC: DIEA = 10: 1: 1.05: 1.02: 1.2: (2~8). The reaction was stirred for 2 hours. When post-treatment was performed using extraction, precipitation, or recrystallization, the yield was <50% and the purity was <80%.

[0054] 3. Boc-GAGAG-OH reacts with HCl·HS-OBn to synthesize Boc-GAGAGS-OBn The Boc-GAGAG-OBn obtained in step (2) above was dissolved in methanol (MeOH), and a catalyst supported on carbon palladium (Pd / C) was added, wherein the equivalent ratio of Boc-GAGAG-OBn: Pd / C = 1: 0.01, the concentration of Boc-GAGAG-OBn in MeOH was 0.1 g / ml, and hydrogen gas was passed through, and the reaction was carried out for 5 hours. After the reaction was completed, the mixture was filtered under reduced pressure, the solvent was evaporated, and then vacuum dried to obtain solid Boc-GAGAG-OH with a yield of over 95% and a purity of over 98%.

[0055] The Boc-GAGAG-OH obtained from the above steps was reacted with HCl·HS-OBn to synthesize Boc-GAGAGS-OBn. The equivalent ratio of each reagent and solvent was DCM: Boc-GAGAS-OH: HCl·HS-OBn: HOBT: EDC: DIEA = 10: 1: 1.05: 1.02: 1.2: (2~8). The reaction was stirred for 2 hours. After the reaction was completed, the yield of this step was <50% and the purity was <80% when post-processing was performed by extraction, precipitation or recrystallization.

[0056] The above results indicate that this amino acid linkage sequence can synthesize the target product, but the yield is low and the purity is generally poor.

[0057] In summary, the amino acid linkage sequence has a decisive impact on the success or failure of the condensation reaction and the quality of the product. This experimental example, by comparing the condensation effects of different linkage sequences, demonstrates that not any arbitrary and reasonable linkage sequence will lead to success in the liquid-phase synthesis of the characteristic hexapeptide GAGAGS of silk fibroin. Compared with linear stepwise extension strategies (such as GAGA+G or GAGAG+S), the "dipeptide + dipeptide" aggregation strategy determined in this invention (i.e., Boc-GA-OH+H-GA-OBn→Boc-GAGA-OBn, with a yield higher than 85% and a purity higher than 96%) can efficiently construct a tetrapeptide intermediate, laying a crucial foundation for the subsequent successful synthesis of the hexapeptide (i.e., Boc-GAGA-OH+HCl·H-GS-OBn→Boc-GAGAGS-OBn), achieving unexpected technical results.

[0058] Experimental Example 2: Screening of Preparation Processes 1. Screening of condensing agents for the synthesis of Boc-GAGA-OBn Following the steps described above, Boc-GA-OH and H-GA-OBn were obtained, and Boc-GAGA-OBn was synthesized. The equivalence ratio of each reagent and solvent was DCM: Boc-GA-OH: H-GA-OBn: HOBT: EDC: DIEA = 10: 1: 1.05: 1.02: 1.5: (2~8). The reaction was stirred for 2 hours. After the reaction, the mixture was extracted twice with saturated NaHCO3 solution, twice with 0.1M KHSO4 aqueous solution, and washed once with saturated sodium chloride aqueous solution. The organic phase was collected, dried with anhydrous Na2SO4, and concentrated under reduced pressure to obtain a pale yellow oily liquid. This indicates that the reaction in the HOBT system was disordered, and attempts to purify it were ineffective, with recrystallization failing to precipitate the product effectively. After purification attempts, the yield was <50% and the purity was <70%. The reaction and related HPLC chromatograms are shown below. Figure 8 As shown.

[0059] 2. Screening of condensing agents for the synthesis of Boc-GAGAGS-OBn (1) DMTMM system: Boc-GAGA-OH and H-GS-OBn after neutralization of excess acid by DIEA were obtained according to the above method. Then, the synthesis conditions of Boc-GAGAGS-OBn were screened. The equivalent ratio of each reagent and solvent was methanol: Boc-GAGA-OH: HCl·H-GS-OBn: DMTMM=10: 1: 1.05: 1.2. The reaction was stirred for 2 hours. After the reaction was completed, the reaction was analyzed. The reaction was disordered and the purity after processing was <50%. The reaction and related HPLC chromatograms are as follows. Figure 9 As shown.

[0060] (2) HOBT / EDC system: Boc-GAGA-OH and H-GS-OBn after neutralization of excess acid with DIEA were obtained according to the above method. Then, the synthesis conditions for Boc-GAGAGS-OBn were screened. The equivalent ratio of each reagent and solvent was DMF: Boc-GAGA-OH: HCl·H-GS-OBn: HOBT: EDC: DIEA = 10: 1: 1.05: 1.05: 1.2: (2~8). The reaction was stirred for 2 hours. After the reaction, the reaction solution was concentrated under reduced pressure, and extracted three times with pure water and DCM at a ratio of 2:1. The DCM layer was collected and extracted twice with saturated NaHCO3 solution, twice with 0.1M KHSO4 aqueous solution, and washed once with saturated sodium chloride aqueous solution. The organic phase was collected, dried with anhydrous Na2SO4, and concentrated under reduced pressure to obtain a pale yellow oily liquid. Analysis of the reaction showed that the reaction was disordered, and recrystallization attempts failed to effectively precipitate the solid, with a purity <50%. The reaction and related HPLC chromatograms are shown below. Figure 10 As shown.

[0061] (3) PFP active intermediate: Boc-GAGA-OH and H-GS-OBn after neutralization of excess acid with DIEA were obtained according to the above method. The obtained Boc-GAGA-OH was dissolved in DCM with PFP and EDC·HCl, where the equivalent ratio of each reagent to solvent was DCM:Boc-GAGA-OH:PFP:EDC·HCl = 10:1.05:1.2. The mixture was stirred for 2 hours. After the reaction, half a volume of water was added to the reaction solution for extraction twice to remove excess EDC·HCl and some water-soluble impurities. The DCM layer was dehydrated using anhydrous sodium sulfate and then evaporated to dryness to obtain Boc-GAGA-PFP. Boc-GAGA-PFP was dissolved in DMF and added to the treated H-GS-OBN, where the equivalent ratio of each reagent to solvent was DMF:Boc-GAGA-PFP:H-GS-OBn = 10:1: 1.1 After the reaction, the reaction solution was concentrated under reduced pressure, and extracted three times with pure water and DCM in a 2:1 ratio. The DCM layer was collected and extracted twice with saturated NaHCO3 solution, twice with 0.1M KHSO4 aqueous solution, and washed once with saturated sodium chloride aqueous solution. The organic phase was collected, dried over anhydrous Na2SO4, and concentrated under reduced pressure to obtain a pale yellow oily liquid. Analysis of the reaction showed a disordered reaction; recrystallization attempts failed to effectively precipitate the solid, with a purity <50%. The reaction and related HPLC chromatograms are shown below. Figure 11 As shown.

[0062] In summary, for the synthesis of Boc-GAGA-OBn and Boc-GAGAGS-OBn, this invention systematically screened various conventional condensing agent systems in the art, such as HOBT / EDC, DMTMM, and PFP active intermediates. However, all these systems resulted in disordered reactions, difficult purification, yields below 50%, and purity below 70%, failing to yield qualified products. In contrast, the preferred condensing agent combination of this invention—using the HOBT / EDC system with methyl tert-butyl ether precipitation in the dipeptide condensation stage, and the HBTU / DIEA system with methanol / ethyl acetate recrystallization in the key condensation stages of tetrapeptide and dipeptide—achieved excellent results with yields >85%, purity >96%, and purity >95%, respectively. This invention reveals that the aforementioned conventional condensing agent systems are completely ineffective in the synthesis of this target peptide; the specific conditions preferred by this invention are the only technical solution that can successfully achieve high yield and high purity preparation among multiple failed pathways, achieving unexpected technical results.

[0063] In summary, this invention provides a liquid-phase synthesis method for silk fibroin peptides. This method, through systematic screening of amino acid linkage sequences and condensing agent systems, ultimately established the optimal technical solution centered on "GA+GA" convergent condensation, combined with specific condensing agent combinations (HOBT / EDC system for dipeptide condensation, and HBTU / DIEA system for the key condensation stages of tetrapeptides and dipeptides) and targeted purification processes (methyl tert-butyl ether precipitation, methanol / ethyl acetate recrystallization). Comparative experiments show that other linkage sequences (such as GAGA+G, GAGAG+S) resulted in yields below 50%, purity below 80%, or even no product formation due to severe steric hindrance or reactivity limitations. Conventional condensing agent systems such as HOBT / EDC, DMTMM, and PFP active ester methods also failed completely in the synthesis of this target peptide, with product purity below 70%. The technical solution established in this invention achieves excellent results with yields >80% and purity >95% (some steps >98%) for all intermediates and final products. This liquid-phase synthesis route is simple to operate, requires no chromatographic purification, and is easy to scale up, providing an economical and efficient technical solution for the high-purity, high-yield, and batch-stable preparation of the characteristic peptide GAGAGS of silk fibroin.

Claims

1. A method for synthesizing silk fibroin peptides, characterized in that: The silk fibroin peptide has the structure shown in formula (I), and the synthesis method is a liquid-phase synthesis method, including the following steps: Formula (I) (a) R 1 -GAGA-OR 2 Remove R 2 Protecting base, to obtain R 1 -GAGA-OH; (b) With amino protecting group as R 3 The glycine and carboxyl protecting groups are R 4 Using serine salt as a raw material, condensation is carried out in the presence of a condensing agent and an organic base to obtain R. 3 -GS-OR 4 ; (c) Take the R obtained in step (b) 3 -GS-OR 4 Remove R 3 Protecting group, yielding H-GS-OR 4 ; (d) Take the R obtained in step (a) 1 -GAGA-OH and H-GS-OR obtained in step (c) 4 Condensation in the presence of a condensing agent and an organic base yields R. 1 -GAGAGS-OR 4 ; (e) Take the R obtained in step (d) 1 -GAGAGS-OR 4 Remove R 4 Protecting base, to obtain R 1 -GAGAGS-OH; (f) Take the R obtained in step (e) 1 -GAGAGS-OH removal R 1 Protecting group, yielding silk fibroin peptide as shown in Formula I; Wherein, the R 1 R 3 R is an amino protecting group. 2 R 4 It is a carboxyl protecting group; In step (d), the condensing agent is benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, and the organic base is N,N-diisopropylethylamine.

2. The method according to claim 1, characterized in that: The R 1 R 3 Each is independently selected from any one of tert-butyloxycarbonyl, benzyloxycarbonyl, or methoxycarbonyl; the R 2 R 4 Each is independently selected from any one of benzyl, methyl, ethyl or tert-butyl.

3. The method according to claim 2, characterized in that: The R 1 R 3 It is tert-butyloxycarbonyl; the R 2 R 4 It is benzyl.

4. The method according to claim 1, characterized in that: In step (a), the R 1 -GAGA-OR 2 The preparation method includes the following steps: (1) With amino protecting group as R 1 The glycine and carboxyl protecting groups are R 2 Using alanine salt as a raw material, condensation is carried out in the presence of a condensing agent and an organic base to obtain R. 1 -GA-OR 2 ; (2) Take the R obtained in step (1) 1 -GA-OR 2 Remove R 2 Protecting base, to obtain R 1 -GA-OH; (3) Take the R obtained in step (1) 1 -GA-OR 2 Remove R 1 Protecting group, yielding H-GA-OR 2 ; (4) Take the R obtained in step (2) 1 -GA-OH reacts with an activating agent and a condensing agent to give an activated intermediate; (5) Combine the activated intermediate obtained in step (4) with the H-GA-OR obtained in step (3). 2 Condensation in the presence of an organic base yields R 1 -GAGA-OR 2 In step (4), the activating agent is pentafluorophenol; the condensing agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. In step (5), the organic base is N,N-diisopropylethylamine.

5. The method according to claim 4, characterized in that: The condensing agent mentioned in steps (1) and (b) is selected from any one or a combination of 1-hydroxybenzotriazole, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate; the organic base is selected from any one of triethylamine, N,N-diisopropylethylamine, or N-methylmorpholine. The removal of R described in steps (2), (a), and (e) 2 Or R 4 Each protecting group is independently prepared using catalytic hydrogenolysis. The removal of R described in steps (3), (c), and (f) 1 Or R 3 Each protecting group is independently prepared using an acidic reagent.

6. The method according to claim 5, characterized in that: In steps (1) and (b), the condensing agent is a combination of 1-hydroxybenzotriazole and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; the organic base is N,N-diisopropylethylamine; In steps (2), (a), and (e), the catalyst in the catalytic hydrogenolysis method is carbon-supported palladium, and the hydrogen source is hydrogen gas. In step (3), the acidic reagent is trifluoroacetic acid; in steps (c) and (f), the acidic reagent is 1,4-dioxane hydrochloride.

7. The method according to claim 6, characterized in that: Each step of the reaction uses an organic solvent independently; In step (1), the organic solvent and the amino protecting group are R. 1 The molar ratio of glycine, alanine salt, 1-hydroxybenzotriazole, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N,N-diisopropylethylamine is (8~12):1:(1.1~1.3):(1.0~1.2):(1.3~1.7):(1.0~1.4); the reaction temperature is 0~10℃ and the time is 1~3h. In step (3), the organic solvent, R 1 -GA-OR 2 The molar ratio of trifluoroacetic acid to trifluoroacetic acid is (8~12):1:(3~10); the reaction time is 1~3 hours. In step (4), the organic solvent, R 1 The molar ratio of GA-OH, pentafluorophenol, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is (8~12): 1: (1.0~1.2): (1.1~1.3); the reaction time is 1~3 h; In step (5), the organic solvent, the activation intermediate, and H-GA-OR 2 The molar ratio of N,N-diisopropylethylamine is (8~12):1:(0.9~1.1):(2~8); the reaction time is 1~3 h; In step (b), the organic solvent and the amino protecting group are R. 3 The molar ratio of glycine, serine, 1-hydroxybenzotriazole, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N,N-diisopropylethylamine is (8~12):1:(1.1~1.3):(1.0~1.2):(1.1~1.3):(1.3~1.7); the reaction temperature is 0~10℃ and the time is 1~3h. In step (c), the organic solvent, R 3 -GS-OR 4 The molar ratio of 1,4-dioxane hydrochloride to 1,4-dioxane is (8-12):1:(3-10); the reaction time is 1-3 hours. In step (d), the organic solvent, R 1 -GAGA-OH、H-GS-OR 4 The molar ratio of benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate and N,N-diisopropylethylamine is (8~12):1:(1.0~1.2):(1.1~1.5):(2~8); the reaction time is 1~3h.

8. The method according to claim 6, characterized in that: Steps (2), (a), and (e) are all carried out under organic solvent conditions. The organic solvent and R... a -peptide-OR b The molar ratio of palladium to carbon-supported palladium is (8~12):1:(0.005~0.02); the reaction time is 3~8 h; the R... a -peptide-OR b For R 1 -GA-OR 2 R 1 -GAGA-OR 2 Or R 1 -GAGAGS-OR 4 .

9. The method according to claim 6, characterized in that: Step (f) is carried out under organic solvent conditions, where the organic solvent and R... 1 The molar ratio of -GAGAGS-OH to 1,4-dioxane hydrochloride is (8~12):1:(3~10); the reaction time is 1~3h.

10. The method according to claim 1, characterized in that: In addition to the deprotection reaction, after each step of the reaction is completed, it also includes any one or more of the following purification steps: extraction, drying with anhydrous sodium sulfate, concentration under reduced pressure, filtration, precipitation, and recrystallization.

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