Fmoc-Tea-OH, preparation method thereof and application of Fmoc-Tea-OH in polypeptide synthesis

Abstract

The invention belongs to the technical field of polypeptide synthesis, and particularly relates to Fmoc-Tea-OH as well as a preparation method and application thereof in polypeptide synthesis. The method comprises the following steps: taking L-theanine and Fmoc-Osu as raw materials, reacting in a weakly alkaline water phase system, and purifying to obtain high-purity Fmoc-Tea-OH. On the basis, an Fmoc solid-phase synthesis strategy is adopted, a condensation system is optimized, accurate coupling of theanine and other amino acids is successfully achieved, and the target polypeptide which is clear in structure and standard in purity is obtained. The problem that the theanine is difficult to participate in solid-phase synthesis of the polypeptide is solved, the application blank of the theanine in the field of polypeptide synthesis is filled, the molecular diversity of the polypeptide for cosmetics is enriched by introducing the unique structure of the theanine, and a foundation is laid for developing a novel skin care component with dual activities of the theanine and the polypeptide.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of polypeptide synthesis, and particularly relates to Fmoc-Tea-OH, a preparation method thereof and application thereof in polypeptide synthesis. BACKGROUND

[0002] As a natural non-protein amino acid, theanine has been widely used in food and health products due to its unique physiological activity (such as stress relief, antioxidant, immune enhancement, etc.), but its research in the field of polypeptides has been in a blank state for a long time. At the same time, as active substances formed by linking amino acids through peptide bonds, polypeptides exhibit significant advantages in the field of cosmetics: such polypeptides usually have low molecular weight (<500 Da), good water solubility and low polarity, and can achieve multiple skin care effects such as moisturizing, antioxidant, anti-wrinkle, whitening, etc., and are the research focus of current functional cosmetic ingredients.

[0003] However, the existing polypeptides for cosmetics are mostly constructed based on natural protein amino acids, and there is a lack of research on introducing theanine into polypeptide sequences, mainly due to the lack of an appropriate synthesis strategy for theanine derivatives - the special structure of theanine makes it difficult to directly participate in conventional solid-phase synthesis reactions.

[0004] Therefore, it is necessary to develop a chemical method that can efficiently introduce theanine into polypeptide sequences, solve the compatibility problem of theanine in polypeptide solid-phase synthesis, and achieve the diversification synthesis of polypeptides containing theanine, which has an urgent need and important significance for enriching the types of polypeptides for cosmetics and improving their overall performance. SUMMARY

[0005] The purpose of the present application is to provide a Fmoc-Tea-OH, a preparation method thereof and application thereof in polypeptide synthesis, which solves the problem that theanine is difficult to participate in polypeptide solid-phase synthesis and realizes the precise coupling of theanine and other amino acids, and finally obtains a target polypeptide with a clear structure and up-to-standard purity.

[0006] In order to achieve the above purpose, the present application provides the following technical solutions: A preparation method of Fmoc-Tea-OH, comprising the following steps: adding saturated sodium bicarbonate aqueous solution to L-theanine, stirring at 200-400 rpm until dissolved, adding Fmoc-Osu in portions under stirring to react, purifying after the reaction is completed, combining single component eluent, and rotary evaporation to obtain white solid crude product, which is Fmoc-Tea-OH.

[0007] Preferably, the solid-liquid ratio of the L-theanine and the saturated sodium bicarbonate aqueous solution is 1g: (15-25) mL.

[0008] By selecting Fmoc-Osu as the amino protecting agent of theanine, the high reactivity and selectivity of Fmoc-Osu to amino group are utilized to realize selective protection, avoid the interference of carboxyl or other groups of theanine to subsequent coupling, and solve the core problem that theanine cannot directly participate in solid-phase synthesis. Meanwhile, saturated sodium bicarbonate aqueous solution is selected as the reaction solvent / alkali system to replace the traditional organic alkali system, which provides a mild weak alkaline environment, can promote the reaction of Fmoc-Osu and amino group, and avoid the racemization or side reaction of theanine that may occur under strong alkaline conditions, thereby ensuring the purity of the product.

[0009] Preferably, the molar ratio of L-theanine and Fmoc-Osu is 1: (1-1.2).

[0010] Preferably, the specific conditions of the divided addition are as follows: divided into 3 times, and each time interval is 8-12 min.

[0011] Preferably, the specific conditions of the reaction are as follows: the temperature is 20-25℃, the reaction time is 20-25 h, and the pH value of the system is maintained at 8.0-8.5.

[0012] The specific steps of the purification are as follows: adjusting the pH value of the system to 3.0-4.0 with citric acid aqueous solution, extracting 2-4 times with ethyl acetate, detecting each extraction with TLC until the L-theanine spot disappears, combining the organic phases, drying, and filtering to obtain the filtrate, and then concentrating to 1 / 10 of the volume of saturated sodium bicarbonate aqueous solution, and then loading a silica gel chromatography column for purification.

[0013] Preferably, the mass fraction of citric acid in the citric acid aqueous solution is 10%.

[0014] Preferably, during the extraction, the addition amount of ethyl acetate for each extraction is 70%-80% of the volume of the saturated sodium bicarbonate aqueous solution.

[0015] Preferably, the specific steps of the extraction are as follows: adding ethyl acetate to the sample system to be treated, oscillating thoroughly, and then standing to separate the layers. After the interface between the two phases is clear, the lower liquid is slowly discharged through the piston of a separatory funnel, and the upper ethyl acetate extract is collected and reserved.

[0016] Preferably, the specific steps of the TLC detection are as follows: taking a silica gel thin layer plate, lightly drawing a baseline parallel to the bottom edge at a distance of 1.5-2 cm from the bottom edge with a pencil, taking an appropriate amount of sample solution with a micro-capillary tube, spotting the sample on the baseline with a diameter controlled within 2-3 mm, and repeating the spotting 1-2 times if the concentration is low after the first spot is dried, taking 5% methanol-dichloromethane developing agent by volume ratio, mixing uniformly, and then pouring into a developing tank, observing and recording the number, position and color of the spots with a 254 nm ultraviolet lamp.

[0017] Preferably, the specific conditions of the drying are as follows: the drying agent is anhydrous sodium sulfate, the addition amount is consistent with the mass of L-theanine, and the drying time is 1.5-2.5 h.

[0018] Preferably, the specific steps of the chromatography purification are as follows: after the concentrated liquid is loaded, an eluent is used for elution.

[0019] Preferably, the specific steps of the chromatography purification are as follows: after the concentrated liquid is loaded, an eluent is used for elution.

[0020] Preferably, the specific conditions of the elution are as follows: the flow rate is 2 mL / min.

[0021] Preferably, the specific conditions of the rotary evaporation are as follows: the water bath temperature is 45℃, the vacuum degree is 0.08-0.09 MPa, and the rotation speed is 50-80 rpm.

[0022] The second aspect of the present application provides Fmoc-Tea-OH prepared by the preparation method of the Fmoc-Tea-OH, and the structural formula of the Fmoc-Tea-OH is shown in Figure 1 .

[0023] The third aspect of the present application provides the application of the Fmoc-Tea-OH in polypeptide synthesis, which is applied to the preparation of linear peptides and cyclic peptides.

[0024] The synthesis method of the linear peptide comprises the following steps: taking resin 1 as a carrier, and coupling amino acids from the C-terminal to the N-terminal according to the amino acid arrangement order of the required linear peptide.

[0025] Preferably, the synthesis method of the linear peptide comprises the following steps: after the resin 1 is swelled, the C-terminal amino acid is fixed on the swelled resin 1, according to the amino acid sequence of the desired linear peptide, the Fmoc protecting group is removed in turn, the resin is washed, the amino acid is coupled, after the synthesis is completed, the peptide chain is cut, and after purification and lyophilization, the final linear peptide chain is obtained, that is, the linear peptide.

[0026] Preferably, the C-terminal amide modification of the resin 1 has a substitution value of 0.5-1.0 mmol / g.

[0027] In some preferred embodiments, the resin 1 is a Rink Amide-AM resin.

[0028] The specific steps of the swelling are as follows: the resin 1 is placed in a T-shaped polypeptide solid-phase synthesis tube, DMF is added, and the shaker is shaken.

[0029] Preferably, in the specific steps of the swelling, the ratio of the resin 1 to DMF is 1 mmol:(40-60) mL.

[0030] Preferably, the specific conditions of the shaker shaking are as follows: the rotation speed is 250-300 rpm, and the time is 10-20 min.

[0031] Preferably, the specific steps of removing the Fmoc protecting group are as follows: the DMF is drained, the deprotection agent is added, the shaker is shaken at 200-250 rpm for 8-12 min, then the deprotection agent is added again, shaken for 20 min, and then drained.

[0032] Preferably, the deprotection agent is any one of a 50% morpholine-DMF mixed solution or a 20% piperidine-DMF solution.

[0033] Preferably, the ratio of the resin 1 to the deprotection agent is 1 mmol:(40-60) mL.

[0034] The specific steps of washing the resin are as follows: the resin is washed with an organic solvent, and then drained for standby.

[0035] The organic solvent includes one or more of DMF, methanol, dichloromethane, and DMF, and the washing frequency of each organic solvent is independently 1-3 times.

[0036] Preferably, the sequence of coupling the amino acid is to sequentially couple from the C-terminal to the N-terminal according to the sequence.

[0037] The coupling of the amino acid is a cyclic process, and the coupling method of all amino acids is consistent. After coupling, the Fmoc protecting group is removed in turn and the resin is washed. After each coupling, the Kaiser reagent is used for detection, and the resin is colorless for complete coupling.

[0038] The specific steps of the coupling amino acid are: dissolving Fmoc-protected amino acid, condensing agent 1 in DMF, adding the swollen resin 1 after ultrasonic dissolution, and shaking on a 200-250 rpm shaker for 1.5-2.5 h.

[0039] Preferably, the Fmoc-protected amino acid includes but is not limited to any one of Fmoc-Arg(Pbf)-OH, Fmoc-Tea-OH, Fmoc-Gln-OH, Fmoc-Glu-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Val-OH, Fmoc-Met-OH, Fmoc-Pal-OH, which are sequentially selected for coupling.

[0040] Preferably, the condensing agent 1 includes HBTU, HOBT, DIPEA, and the molar ratio is 1:1:2.

[0041] Preferably, the molar ratio of the Fmoc-protected amino acid to the condensing agent 1 is 1:4.

[0042] Preferably, the ratio of the total molar amount of the Fmoc-protected amino acid and the condensing agent 1 to the molar amount of the resin 1 is (15-25):1.

[0043] The selection of HBTU, HOBt, and DIPEA as condensing agents, and the control of their molar ratio and the molar ratio with Fmoc-protected amino acid, can improve the coupling efficiency of amino acid, effectively overcome the incomplete coupling problem caused by the steric hindrance or reactivity of amino acid, and ensure the nearly quantitative completion of each step reaction. HBTU, as a high-efficiency uranium salt condensing agent, can quickly convert the carboxyl group of amino acid into a highly active ester intermediate, providing power for the formation of peptide bond, significantly reducing the activation energy of subsequent reaction with resin amino, and helping to react with the carboxyl group of tea amino acid with low reactivity. However, the high activity of the activation may lead to racemization of the chiral center of the amino acid, generating impurities, and damaging the purity and biological activity of the final polypeptide product. The addition of HOBt serves as an auxiliary ligand for this activation step, and also timely captures possible by-products to inhibit racemization side reactions. The addition of double amount of DIPEA provides an alkaline environment, which not only promotes the deprotonation of carboxyl group, but also neutralizes the protons released during the reaction and acidic by-products, maintaining a neutral to alkaline environment in the system. Excessive DIPEA can also inhibit the self-cyclization of tea amino acid amino and other side reactions. Through the design of molar ratio, efficient, stable and low side reaction coupling of tea amino acid and conventional amino acid is finally achieved.

[0044] Preferably, in the specific steps of the coupling amino acid, the ratio of resin 1 to DMF is 1 mmol:(40-60) mL.

[0045] Preferably, the specific conditions of the ultrasonic dissolution are as follows: ultrasonic time is 1-3 min, ultrasonic power is 240 W, ultrasonic temperature is 25℃, and ultrasonic frequency is 25 kHz.

[0046] The specific steps of the cutting peptide chain are as follows: after the synthesis is completed, the resin is washed with DMF and methanol for 3 times in sequence and dried under vacuum, the cutting solution is added, the shaking is performed at 180-220 rpm for 1-2 h, the cutting solution is collected and the resin is washed, and the nitrogen blowing is performed to dry the volume to 1 / 8 of the cutting solution volume to obtain the crude peptide chain.

[0047] Preferably, the specific conditions of the vacuum drying are as follows: vacuum degree is 5-10 Pa, temperature is 25℃, and time is 1 h.

[0048] Preferably, the cutting solution comprises TFA, Tis and water, and the mass ratio is 95:2.5:2.5.

[0049] Preferably, the ratio of the resin 1 and the cutting solution is 1 mmol:(70-90) mL.

[0050] In some preferred schemes, the step of cutting the peptide chain can be repeated for 1 time, and the two cutting solutions are combined and then dried under nitrogen blowing.

[0051] The specific steps of the purification and freeze-drying are as follows: the crude peptide chain is added with ether at -20℃, the precipitation is performed for 15 min, the centrifugation is performed, the supernatant is discarded, the precipitate is dissolved with a trifluoroacetic acid-dichloromethane solution with a mass fraction of 0.1% trifluoroacetic acid, the reverse phase HPLC purification is performed, the main peak is collected, and the freeze-drying is performed to obtain a white powder.

[0052] Preferably, the ratio of the resin 1 and the ether is 1 mmol:(350-450) mL.

[0053] Preferably, the specific conditions of the centrifugation are as follows: the rotation speed is 4000 rpm, and the time is 10 min.

[0054] Preferably, the specific steps of the reverse HPLC purification are as follows: ① Sample pretreatment: Dissolve the sample to be purified in the initial mobile phase, controlling the concentration at 5-20 mg / mL to ensure complete dissolution. Filter the sample solution using a 0.22 μm organic phase filter membrane to remove particulate impurities and prevent column clogging. ② Chromatographic system preparation: Select a reversed-phase column (commonly a C18 column, 4.6 × 250 mm, 5 μm particle size), prepare the mobile phase, degas the mobile phase by sonication for 15 minutes, and filter it using a 0.22 μm filter membrane. Turn on the HPLC system, set the column temperature to 35℃, connect the column, and equilibrate the system with the initial mobile phase until the baseline is stable. ③ Chromatographic condition settings: Flow rate is 1.0 mL / min, detection wavelength: selected according to the UV absorption characteristics of the target compound (e.g., 254 nm, 210 nm, etc.). Gradient elution program: Set according to previous analysis results, for example: 0-5 min, 5% organic phase; 5-30 min, 5%-95% organic phase; 30-35 min, 95% organic phase. ④ Sample injection and separation: Draw an appropriate amount of sample solution with the injection needle, inject the sample, start the program, and record the chromatogram. Observe the chromatographic peaks and determine the target peak position based on the retention time. When the target peak appears, open the fraction collector to collect the corresponding eluent. ⑤ Post-processing: Combine the collected liquids with the same target component, remove the solvent by rotary evaporation to obtain the purified sample, and perform HPLC purity detection on the purified product.

[0055] Preferably, the mobile phase includes mobile phase A and mobile phase B, where mobile phase A is a TFA aqueous solution with a TFA mass fraction of 0.1% and mobile phase B is a TFA-acetonitrile solution with a TFA mass fraction of 0.1%.

[0056] Preferably, the organic phase is mobile phase B, and 5% of the organic phase is a mixture of 5% mobile phase B and 95% mobile phase A.

[0057] Preferably, the specific conditions for freeze-drying are: temperature -80℃ and time 24h.

[0058] The method for synthesizing the cyclic peptide includes the following steps: using resin 2 as a carrier, a linear peptide containing theanine is first synthesized, and then intramolecularly cyclized to obtain the peptide.

[0059] Preferably, the method for synthesizing the cyclic peptide includes the following steps: after swelling resin 2, loading the first amino acid, sequentially coupling amino acids from the C-terminus to the N-terminus of the cyclic peptide sequence to synthesize a linear peptide chain, and then sequentially performing intramolecular cyclization, cleavage and purification, followed by freeze drying to obtain the final product.

[0060] Preferably, the substitution value of resin 2 is 0.3-0.7 mmol / g.

[0061] In some preferred embodiments, the resin 2 is 2-CTC Resin resin.

[0062] Preferably, the swelling step of resin 2 is consistent with the specific steps of the swelling of resin 1.

[0063] Resins are selected differently based on the structure of the target peptides. For linear peptides, Rink Amide-AM resin is used. This is to directly obtain C-terminal amidated peptides, which more closely mimics the state of natural peptides, and simplifies the synthesis process by eliminating the need for additional amidation reagents. For cyclic peptides, which require linear synthesis followed by cyclization, 2-CTC Resin resin is selected. This resin is characterized by easy cleavage and high amino acid loading efficiency, facilitating subsequent cleavage, and eliminates the need for side-chain protecting groups, simplifying pre-cyclization treatment.

[0064] The specific steps for loading the first amino acid are similar to those for coupling the amino acid described above. Specifically, the DMF is dried, Fmoc-protected amino acid and condensing agent 2 are dissolved in the DMF, dissolved by ultrasonication, and then the swollen resin 2 is added. The mixture is shaken on a shaker at 200-250 rpm for 1.5-2.5 hours. The resin is then washed sequentially to remove any unreacted reagents.

[0065] Preferably, the condensing agent 2 is DIPEA.

[0066] Preferably, the molar ratio of Fmoc-protected amino acid to condensing agent 2 is 1:1.

[0067] Preferably, the ratio of the total molar amount of Fmoc-protected amino acid and condensing agent 2 to the molar amount of resin 1 is (5-10):1.

[0068] The specific steps for washing the resin are the same as those for washing the resin in the above-mentioned linear peptide synthesis process.

[0069] In the method for synthesizing the cyclic peptide, the specific steps of amino acid coupling are as follows: first, deprotection is performed, and then a coupling reaction is carried out.

[0070] The specific steps for deprotection are the steps of removing the Fmoc protecting group and washing the resin in the above-mentioned linear peptide synthesis process.

[0071] The specific steps of the coupling reaction are the same as the amino acid coupling step in the above-mentioned linear peptide synthesis process. The difference is that resin 1 in the amino acid coupling step in the above-mentioned linear peptide synthesis process is replaced with resin 2. After the coupling is completed, the Kaiser reagent is used for detection. If the resin is colorless, it indicates that the coupling is complete; if it is blue or purple-red, the coupling step needs to be repeated.

[0072] The specific steps of the intramolecular cyclization are as follows: first, N-terminal deprotection is performed, and then a cyclization reaction is carried out.

[0073] The specific steps for N-terminal deprotection are the steps of removing the Fmoc protecting group and washing the resin in the above-mentioned linear peptide synthesis process.

[0074] The specific steps of the cyclization reaction are as follows: add DMF aqueous solution, then add condensing agent 3, shake on a shaker at 200-250 rpm for 8-12 hours, and it is ready.

[0075] Preferably, the mass fraction of DMF in the DMF aqueous solution is 50%.

[0076] Preferably, the ratio of resin 2 to DMF aqueous solution is 1 mmol: (80-120) mL.

[0077] Intramolecular cyclization in DMF aqueous solution significantly reduced peptide chain concentration, effectively inhibiting intermolecular polymerization side reactions and significantly improving the selectivity and yield of intramolecular head-tail cyclization. This resulted in the successful synthesis of structurally well-defined theanine-containing cyclic peptides, filling a gap in the synthesis of theanine-containing cyclic peptides.

[0078] Preferably, the condensing agent 3 includes HBTU, HOBT, and DIPEA in a molar ratio of 1:1:2.

[0079] Preferably, the molar ratio of resin 2 to condensing agent 3 is 1:12.

[0080] The specific steps for cyclic peptide cleavage and purification are consistent with the specific steps for peptide chain cleavage, purification, and lyophilization in the above-mentioned linear peptide synthesis process.

[0081] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: 1. This invention provides a method for preparing Fmoc-Tea-OH, using L-theanine and Fmoc-Osu as raw materials, reacting them in a weakly alkaline aqueous system, and purifying them to obtain high-purity Fmoc-Tea-OH. Based on this, a solid-phase synthesis strategy using Fmoc is employed, and the condensation system is optimized to successfully achieve precise coupling of theanine with other amino acids, obtaining a target polypeptide with a well-defined structure and meeting purity standards.

[0082] 2. This invention solves the problem of theanine's difficulty in participating in solid-phase peptide synthesis, not only filling the gap in the application of theanine in the field of peptide synthesis, but also enriching the molecular diversity of peptides for cosmetics by introducing the unique structure of theanine, laying the foundation for the development of novel skin care ingredients with both theanine and peptide activities.

[0083] 3. This invention solves the technical bottleneck that theanine is difficult to directly participate in the solid-phase synthesis of peptides, and provides a suitable synthesis scheme for theanine derivative Fmoc-Tea-OH, which breaks through its compatibility limitations in peptide sequence construction and realizes the efficient coupling of theanine with other amino acids.

[0084] 4. This invention addresses the shortcomings of existing cosmetic peptides in terms of synergistic activity. It utilizes the synergistic effect of the natural activity of theanine (such as anti-oxidation and soothing) and the skin care effects of peptides (such as moisturizing and anti-wrinkle) to enhance the overall skin care performance of peptides and solve the problem of traditional peptides having single functions and limited effects.

[0085] 5. This invention utilizes Fmoc-Osu to specifically protect the α-amino group of theanine, generating Fmoc-Tea-OH. The reaction is carried out in a weakly alkaline environment, taking advantage of the high reactivity of Fmoc-Osu with the amino group to achieve selective protection and avoid interference from the carboxyl group or other groups of theanine in subsequent coupling.

[0086] 6. This invention employs the Fmoc solid-phase synthesis method, using resin as a carrier, to progressively couple amino acids from the C-terminus to the N-terminus, ultimately obtaining a linear peptide chain. The linear peptide synthesis method described in this invention allows for precise sequence control, and theanine achieves efficient coupling with other amino acids via Fmoc-Tea-OH.

[0087] 7. This invention prepares cyclic peptides through a process of "first synthesizing linear peptides, then intramolecular cyclization." A specific resin is selected, and a linear peptide chain containing theanine is first synthesized. After removing the N-terminal Fmoc, a condensing agent is used in a diluted DMF solution to promote the cyclization of the C-terminal carboxyl group and the N-terminal amino group, forming a stable cyclic structure. The cyclic peptide contains one or two theanine molecules, and the cyclic structure enhances its resistance to enzymatic degradation. Attached Figure Description

[0088] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0089] Figure 1 The structural formula of Fmoc-Tea-OH prepared in this invention is shown below; Figure 2 The hydrogen spectrum of Fmoc-Tea-OH prepared in this invention is shown below. Figure 3 The HPLC chromatogram of the linear peptide prepared in Example 2 of this invention; Figure 4 The HPLC chromatogram of the linear peptide prepared in Example 3 of this invention; Figure 5 The HPLC chromatogram of the linear peptide prepared in Example 4 of this invention; Figure 6 The HPLC chromatogram of the linear peptide prepared in Example 5 of this invention; Figure 7 The HPLC chromatogram of the linear peptide prepared in Example 6 of this invention; Figure 8 The HPLC chromatogram of the linear peptide prepared in Example 7 of this invention; Figure 9 The HPLC chromatogram of the cyclic peptide prepared in Example 8 of this invention; Figure 10 This is the HPLC chromatogram of the cyclic peptide prepared in Example 9 of the present invention. Detailed Implementation

[0090] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0091] All raw materials used in this invention are commercially available, specifically: Resin 1 is a Rink Amide-AM resin with C-terminal amidation modification and a substitution value of 0.7 mmol / g, derived from Maclean.

[0092] Fmoc-protected amino acids are all derived from Maclean's.

[0093] Resin 2 is a 2-CTC Resin resin with a substitution value of 0.5 mmol / g, derived from Maclean.

[0094] The silica gel chromatography column had a mesh size of 200-300 and was sourced from Maclean's.

[0095] Example 1

[0096] This embodiment provides a method for preparing Fmoc-Tea-OH, which includes the following steps: 10.0 g of L-theanine (54.3 mmol) is added to 200 mL of saturated sodium bicarbonate aqueous solution, and stirred at 300 rpm until dissolved (pH approximately 8.3). Fmoc-Osu is then added in portions while stirring to carry out the reaction. After the reaction is complete, the mixture is purified, and the eluents of the single components are combined and rotary evaporated to obtain a white solid crude product, which is Fmoc-Tea-OH.

[0097] The specific conditions for adding the ingredients in stages are: divide the ingredients into 3 equal parts, with an interval of 10 minutes between each addition.

[0098] The specific conditions for the reaction are: temperature of 23℃, reaction time of 24h, and maintaining the pH of the system at 8.0-8.5.

[0099] The specific purification steps are as follows: adjust the pH of the system to 3.5 with a 10% citric acid aqueous solution, extract three times with 150 mL of ethyl acetate, and detect each extraction by TLC until the L-theanine spot disappears. Combine the organic phases, dry, filter to obtain the filtrate, concentrate by rotary evaporation to 1 / 10 of the volume of saturated sodium bicarbonate aqueous solution, and then purify by silica gel chromatography.

[0100] The specific steps of the extraction are as follows: Add ethyl acetate to the sample system to be treated, shake thoroughly, and then allow it to stand to separate into layers. After the interface between the two phases is clear, slowly release the lower layer liquid through the stopcock of the separatory funnel, and collect and retain the upper layer of ethyl acetate extract.

[0101] The specific steps of the TLC detection are as follows: Take a silica gel thin-layer plate and lightly draw a baseline parallel to the bottom edge with a pencil at a distance of 1.5-2 cm from the bottom edge. Use a microcapillary tube to draw an appropriate amount of sample solution and spot it on the baseline. The spot diameter should be controlled within 2-3 mm. If the concentration is low, repeat the spotting 1-2 times after the first spot dries. Measure dichloromethane and methanol at a volume ratio of 95:5, mix them evenly to prepare a 5% methanol-dichloromethane developing solvent, pour it into the developing tank, and observe and record the number, position, and color of the spots using a 254 nm UV lamp. Measure the distance from the center of each spot to the baseline and the distance from the front of the developing solvent to the baseline, and calculate the Rf value of each spot.

[0102] The specific drying conditions are as follows: the desiccant is anhydrous sodium sulfate, the amount added is consistent with the mass of L-theanine, and the drying time is 2 hours.

[0103] The specific steps of the chromatography purification are as follows: After loading the concentrated liquid onto the sample, elute with an eluent. According to the properties of the target compound, prepare a series of methanol-dichloromethane mixed eluents with gradient concentrations in advance (starting from pure dichloromethane, gradually increasing the proportion of methanol, with the methanol mass fraction from 5%, 10%, 20%... until pure methanol). Each concentration of eluent needs to be thoroughly mixed and ultrasonically degassed for 10 minutes, and then allowed to stand for later use.

[0104] The elution flow rate is 2 mL / min.

[0105] The specific conditions for the rotary evaporation are: water bath temperature of 45℃, vacuum degree of 0.085MPa, and rotation speed of 60rpm.

[0106] The chemical structural formula of the prepared Fmoc-Tea-OH is as follows: ; Fmoc-Tea-OH proton spectrum, see Figure 2: ¹H NMR, MeOH-d4, 500 MHz δ 7.92 (d, J=7.5 Hz, 2H), 7.74 (d, J=7.2 Hz, 2H), 7.41 (t, J=7.3 Hz, 2H), 7.32 (t, J=7.5 Hz, 2H), 6.85 (s, 1H), 6.72 (s, 1H), 4.38 (d, J=6.8 Hz), 4.21 (t, J=6.8 Hz, 1H), 3.75 (m, 1H), 2.31 (t, J=7.2 Hz), 1.72 (m, 2H).

[0107] The purity of the prepared Fmoc-Tea-OH was 95.2%, and the yield was 88.9%.

[0108] Example 2 This embodiment provides a theanine-containing linear peptide with the amino acid sequence Glu-Glu-Tea-Gln-Arg-Arg-NH2. The synthesis method is as follows: 0.1 mmol (about 143 mg) of resin 1 is swollen, and the C-terminal amino acid is fixed on the swollen resin 1. According to the desired amino acid sequence of the linear peptide, the Fmoc protecting group is removed, the resin is washed, and the coupled amino acids are removed in sequence. After synthesis, the peptide chain is cut, purified and lyophilized to obtain the final linear peptide chain, which is the linear peptide.

[0109] The specific steps for swelling are as follows: place resin 1 in a T-shaped polypeptide solid-phase synthesis tube, add 5 mL of DMF, and shake on a shaker.

[0110] The specific conditions for the shaking table oscillation are: a rotation speed of 280 rpm and a time of 15 min.

[0111] The specific steps for removing the Fmoc protecting group are as follows: DMF is dried, 5 mL of deprotecting agent is added, and the mixture is shaken on a shaker at 220 rpm for 10 min. Then, the mixture is dried again, 5 mL of deprotecting agent is added, and the mixture is shaken for 20 min.

[0112] The deprotectant is a piperidine-DMF solution with a piperidine mass fraction of 20%.

[0113] The specific steps for washing the resin are as follows: wash the resin alternately with DMF (3 times, 5 mL each time) and DCM (3 times, 5 mL each time), and then dry it for later use.

[0114] The amino acids are coupled sequentially from the C-terminus to the N-terminus. In this embodiment, the amino acid sequence of the straight-chain peptide is Glu-Glu-Tea-Gln-Arg-Arg-NH2. Arg is coupled in the first round, and then Arg, Gln, Tea (using Fmoc-Tea-OH prepared in Example 1), Glu, and Glu are coupled sequentially from the C-terminus to the N-terminus.

[0115] The coupling of amino acids is a cyclic process, and the coupling method for all amino acids is the same. After coupling, the Fmoc protecting group must be removed and the resin washed in sequence. After each coupling, the resin is detected with Kaiser reagent. The coupling is complete when the resin is colorless.

[0116] Taking the first round of Arg coupling as an example, the specific steps for coupling amino acids are as follows: dissolve 0.4 mmol Fmoc-Arg(Pbf)-OH and 1.6 mmol condensing agent 1 in 5 mL DMF, dissolve by sonication, add the swollen resin 1, and shake on a shaker at 220 rpm for 2 hours.

[0117] The condensing agent 1 is HBTU, HOBT, and DIPEA in a molar ratio of 1:1:2.

[0118] The specific conditions for ultrasonic dissolution are as follows: ultrasonic time is 2 minutes, ultrasonic power is 240W, ultrasonic temperature is 25℃, and ultrasonic frequency is 25kHz.

[0119] The specific steps for cleaving the peptide chain are as follows: After synthesis, the resin is washed sequentially with DMF (3 times, 5 mL each time) and methanol (3 times, 5 mL each time) and dried under vacuum. 8 mL of cleaving solution is added, and the mixture is shaken at 200 rpm for 2 h. The cleaving solution is collected and the resin is rinsed. The cleavage is repeated once, and the two cleaving solutions are combined and dried with nitrogen to 1 mL to obtain the crude peptide chain.

[0120] The specific conditions for vacuum drying are: vacuum degree of 7.5 Pa, temperature of 25℃, and time of 1 hour.

[0121] The cutting fluid comprises TFA, Tis, and water in a mass ratio of 95:2.5:2.5.

[0122] The specific steps of purification and freeze-drying are as follows: 40 mL of diethyl ether at -20℃ is added to the crude peptide chain, and after precipitation for 15 min, the mixture is centrifuged, the supernatant is discarded, the precipitate is dissolved in 5 mL of trifluoroacetic acid-dichloromethane solution with a mass fraction of 0.1%, purified by reversed-phase HPLC, the main peak is collected, and the mixture is freeze-dried to obtain a white powder.

[0123] The specific conditions for centrifugation are: a rotation speed of 4000 rpm and a time of 10 min.

[0124] The specific steps of the reverse HPLC purification are as follows: ① Sample pretreatment: Dissolve the sample to be purified in the initial mobile phase, controlling the concentration at 15 mg / mL, ensuring complete dissolution. Filter the sample solution using a 0.22 μm organic phase filter membrane to remove particulate impurities and prevent column clogging. ② Chromatographic system preparation: Select a C18 reversed-phase column with dimensions of 4.6 × 250 mm and a particle size of 5 μm. Prepare the mobile phase, degas it by sonication for 15 minutes, and filter it using a 0.22 μm filter membrane. Turn on the HPLC system, set the column temperature to 35℃, connect the column, and equilibrate the system with the initial mobile phase until the baseline is stable. ③ Chromatographic conditions: Flow rate is 1.0 mL / min, detection wavelengths are 254 nm and 210 nm. Gradient elution program: 0-5 min, 5% organic phase; 5-30 min, 5%-95% organic phase; 30-35 min, 95% organic phase. ④ Sample injection and separation: Draw 30 μL of sample solution into the syringe, inject the sample, start the program, record the chromatogram, observe the elution of the chromatographic peaks, and determine the position of the target peak based on the retention time. When the target peak appears, start the fraction collector to collect the corresponding eluent. ⑤ Post-processing: Combine the collected liquids with the same target component, remove the solvent by rotary evaporation, and obtain the purified sample. Perform HPLC purity detection on the purified product.

[0125] The mobile phase consists of mobile phase A and mobile phase B. Mobile phase A is a TFA aqueous solution with a TFA mass fraction of 0.1%, and mobile phase B is a TFA-acetonitrile solution with a TFA mass fraction of 0.1%.

[0126] The specific conditions for freeze-drying are: temperature -80℃ and time 24h.

[0127] The prepared theanine-containing linear peptide has the amino acid sequence Glu-Glu-Tea-Gln-Arg-Arg-NH2 and its structural formula is as follows: ; HPLC detection results are shown below. Figure 3 .from Figure 3 The mass spectrometry results show that the purity of the prepared linear peptide is 97.5%, and the measured molecular weight is 872.75 (the theoretical molecular weight of the target peptide is 871.76).

[0128] Example 3 This embodiment provides a linear peptide containing theanine, with the amino acid sequence Gly-Pro-Gln-Tea-Pro-Gln. Its synthesis method is the same as in Example 2, except that the required Fmoc-protecting amino acids are selected and coupled sequentially according to the amino acid sequence.

[0129] The prepared theanine-containing linear peptide has the amino acid sequence Gly-Pro-Gln-Tea-Pro-Gln, and its structural formula is as follows: ; HPLC detection results are shown below. Figure 4 .from Figure 4 The mass spectrometry results show that the purity of the prepared linear peptide is 95.9%, and the measured molecular weight is 682.76 (the theoretical molecular weight of the target peptide is 681.75).

[0130] Example 4 This embodiment provides a linear peptide containing theanine, with the amino acid sequence Phe-Val-Tea-Pro-Phe-Pro. Its synthesis method is the same as in Example 2, except that the required Fmoc-protecting amino acids are selected and coupled sequentially according to the amino acid sequence.

[0131] The prepared theanine-containing linear peptide has the amino acid sequence Phe-Val-Tea-Pro-Phe-Pro and its structural formula is as follows: ;

[0132] HPLC detection results are shown below. Figure 5 .from Figure 5 The mass spectrometry results show that the purity of the prepared linear peptide is 97.2%, and the measured molecular weight is 762.90 (the theoretical molecular weight of the target peptide is 761.92).

[0133] Example 5 This embodiment provides a linear peptide containing theanine, with the amino acid sequence Glu-Glu-Met-Gln-Arg-Arg-Tea. Its synthesis method is the same as in Example 2, except that the required Fmoc-protecting amino acids are selected and coupled sequentially according to the amino acid sequence.

[0134] The prepared theanine-containing linear peptide has the amino acid sequence Glu-Glu-Met-Gln-Arg-Arg-Tea, and its structural formula is as follows: ; HPLC detection results are shown below. Figure 6 .from Figure 6 The mass spectrometry results show that the purity of the prepared linear peptide is 99.0%, and the measured molecular weight is 1005.79 (the theoretical molecular weight of the target peptide is 1004.13).

[0135] Example 6 This embodiment provides a linear peptide containing theanine, with the amino acid sequence Pal-Val-Gly-Val-Tea-Pro-Gly. Its synthesis method is the same as in Example 2, except that the required Fmoc-protecting amino acids are selected and coupled sequentially according to the amino acid sequence.

[0136] The prepared theanine-containing linear peptide has the amino acid sequence Pal-Val-Gly-Val-Tea-Pro-Gly, and its structural formula is as follows: ; HPLC detection results are shown below. Figure 7 .from Figure 7 The mass spectrometry results show that the purity of the prepared linear peptide is 96.7%, and the measured molecular weight is 823.09 (the theoretical molecular weight of the target peptide is 822.10).

[0137] Example 7 This embodiment provides a linear peptide containing theanine, with the amino acid sequence Gly-Pro-Arg-Pro-Tea-NH2. Its synthesis method is the same as in Example 2, except that the required Fmoc-protecting amino acids are selected and coupled sequentially according to the amino acid sequence.

[0138] The prepared theanine-containing linear peptide has the amino acid sequence Gly-Pro-Arg-Pro-Tea-NH2 and its structural formula is as follows: ; HPLC detection results are shown below. Figure 8 .from Figure 8 The mass spectrometry results show that the purity of the prepared linear peptide is 98.4%, and the measured molecular weight is 581.76 (the theoretical molecular weight of the target peptide is 581.68).

[0139] Example 8 This embodiment provides a theanine-containing cyclopeptide cyclo(Tea-Pro-Gln-Tea-Pro-Gln), with the amino acid sequence Tea-Pro-Gln-Tea-Pro-Gln. The synthesis method is as follows: 0.1 mmol (about 200 mg) of resin 2 is swollen, the first amino acid is loaded, and amino acids are coupled sequentially from the C-terminus to the N-terminus of the cyclopeptide sequence to synthesize a linear peptide chain. Then, intramolecular cyclization, cleavage and purification are performed sequentially, and the product is obtained after lyophilization.

[0140] The swelling steps of resin 2 are the same as the specific steps of resin 1 swelling in Example 2.

[0141] The specific steps for loading the first amino acid are similar to those for coupling amino acids in Example 2. Specifically, the DMF is dried, and 0.4 mmol Fmoc-Gln-OH and 0.4 mmol condensing agent 2 are dissolved in 5 mL of DMF. After ultrasonic dissolution, the swollen resin 2 is added, and the mixture is shaken on a shaker at 220 rpm for 2 hours. The resin is then washed with DMF (3 times, 5 mL each time) and DCM (3 times, 5 mL each time) to remove unreacted reagents.

[0142] The condensing agent 2 is DIPEA.

[0143] In the method for synthesizing the cyclic peptide, the specific steps of amino acid coupling are as follows: first, deprotection is performed, and then a coupling reaction is carried out.

[0144] The specific steps for deprotection are the steps of removing the Fmoc protecting group and washing the resin in the linear peptide synthesis process of Example 2.

[0145] The specific steps of the coupling reaction are the amino acid coupling steps in the linear peptide synthesis process of Example 2. After coupling is completed, the resin is tested with Kaiser's reagent. If the resin is colorless, the coupling is complete; if it is blue or purplish-red, the coupling step needs to be repeated.

[0146] The specific steps of the intramolecular cyclization are as follows: first, N-terminal deprotection is performed, and then a cyclization reaction is carried out.

[0147] The specific steps for N-terminal deprotection are the steps of removing the Fmoc protecting group and washing the resin in the linear peptide synthesis process of Example 2.

[0148] The specific steps of the cyclization reaction are as follows: add 10 mL of DMF aqueous solution, then add 1.2 mmol of condensing agent 3, shake on a shaker at 220 rpm for 10 hours, and it is ready.

[0149] The mass fraction of DMF in the DMF aqueous solution is 50%.

[0150] The condensing agent 3 is HBTU, HOBT, and DIPEA, with a molar ratio of 1:1:2.

[0151] The specific steps for cleavage and purification of the cyclic peptide are consistent with the specific steps for cleavage, purification, and freeze-drying of the linear peptide in Example 2.

[0152] The prepared theanine-containing cyclopeptide (Tea-Pro-Gln-Tea-Pro-Gln) has the following structural formula: ; HPLC detection results are shown below. Figure 9 .from Figure 9The mass spectrometry results show that the purity of the prepared cyclic peptide is 95.2% and the molecular weight is 763.85 (the theoretical molecular weight of the target peptide is 762.5%).

[0153] Example 9 This embodiment provides a theanine-containing cyclo peptide (Phe-Val-Tea-Pro-Phe-Pro), with the amino acid sequence Phe-Val-Tea-Pro-Phe-Pro. Its synthesis method is the same as in Example 8, except that the required Fmoc-protecting amino acids are selected and coupled sequentially according to the amino acid sequence.

[0154] The prepared theanine-containing cyclopeptide (Phe-Val-Tea-Pro-Phe-Pro) has the following structural formula: ; HPLC detection results are shown below. Figure 10 .from Figure 10 The mass spectrometry results show that the purity of the prepared cyclic peptide is 98.9%, and the measured molecular weight is 744.76 (the theoretical molecular weight of the target peptide is 743.91).

[0155] Therefore, the high-purity Fmoc-Tea-OH prepared using the raw materials and methods described in this application can be used to synthesize various linear and cyclic peptides containing theanine. All target peptides have well-defined structures and meet purity standards. This invention solves the problem of theanine's difficulty in participating in solid-phase peptide synthesis, not only filling the application gap of theanine in peptide synthesis but also enriching the molecular diversity of cosmetic peptides by introducing the unique structure of theanine, laying the foundation for developing novel skincare ingredients with both theanine and peptide activities.

[0156] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing Fmoc-Tea-OH, characterized in that, Includes the following steps: Add saturated sodium bicarbonate aqueous solution to L-theanine and stir at 200-400 rpm until dissolved. Add Fmoc-Osu in portions while stirring to react. After the reaction is complete, purify and combine the eluents of the single components. Rotary evaporation yields a white solid crude product, which is Fmoc-Tea-OH. The solid-liquid ratio of the L-theanine and the saturated sodium bicarbonate aqueous solution is 1 g: (15-25) mL; The molar ratio of L-theanine to Fmoc-Osu is 1:(1-1.2).

2. The method for preparing Fmoc-Tea-OH according to claim 1, characterized in that, The specific conditions for the reaction are: temperature of 20-25℃, reaction time of 20-25h, and maintaining the pH of the system at 8.0-8.

5.

3. The method for preparing Fmoc-Tea-OH according to claim 1, characterized in that, The specific purification steps are as follows: adjust the pH of the system to 3.0-4.0 with citric acid aqueous solution, extract with ethyl acetate 2-4 times, and use TLC detection for each extraction until the L-theanine spot disappears. Combine the organic phases, dry, filter to obtain filtrate, concentrate by rotary evaporation to 1 / 10 of the volume of saturated sodium bicarbonate aqueous solution, and then purify by silica gel column chromatography.

4. Fmoc-Tea-OH prepared by the method according to any one of claims 1 to 3.

5. An application of Fmoc-Tea-OH according to claim 4 in polypeptide synthesis, characterized in that, It is used to prepare linear peptides and cyclic peptides.

6. The application of Fmoc-Tea-OH in polypeptide synthesis according to claim 5, characterized in that, The method for synthesizing the linear peptide includes the following steps: using resin 1 as a carrier, amino acids are gradually coupled from the C-terminus to the N-terminus according to the amino acid sequence of the desired linear peptide.

7. The application of Fmoc-Tea-OH in polypeptide synthesis according to claim 6, characterized in that, The method for synthesizing the linear peptide includes the following steps: after swelling resin 1, the C-terminal amino acid is fixed on the swollen resin 1; according to the amino acid sequence of the desired linear peptide, the Fmoc protecting group is removed, the resin is washed, and the coupled amino acid is coupled in sequence; after synthesis, the peptide chain is cut, purified, and lyophilized to obtain the final linear peptide chain, which is the linear peptide.

8. The application of Fmoc-Tea-OH in polypeptide synthesis according to claim 7, characterized in that, The resin 1 is subjected to C-terminal amidation modification with a substitution value of 0.5-1.0 mmol / g.

9. The application of Fmoc-Tea-OH in polypeptide synthesis according to claim 7, characterized in that, The specific steps for the coupled amino acid are as follows: Fmoc-protected amino acid and condensing agent 1 are dissolved in DMF, ultrasonically dissolved, and then the swollen resin 1 is added. The mixture is shaken on a shaker at 200-250 rpm for 1.5-2.5 hours.

10. The application of Fmoc-Tea-OH in polypeptide synthesis according to claim 9, characterized in that, The Fmoc-protected amino acids include, but are not limited to, any one of Fmoc-Arg(Pbf)-OH, Fmoc-Tea-OH, Fmoc-Gln-OH, Fmoc-Glu-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Val-OH, Fmoc-Met-OH, and Fmoc-Pal-OH, which are selected and coupled sequentially.

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