Fmoc-tea-oh, its preparation method and application in polypeptide synthesis

Abstract

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. L-theanine and Fmoc-Osu are used as raw materials to react in a weak alkaline aqueous system, and high-purity Fmoc-Tea-OH is obtained through purification. On this basis, Fmoc solid-phase synthesis strategy is adopted, and the condensation system is optimized, so that precise coupling of theanine and other amino acids is successfully realized, and a target polypeptide with a clear structure and up-to-standard purity is obtained. The application solves the problem that theanine is difficult to participate in polypeptide solid-phase synthesis, not only fills the application blank of theanine in the field of polypeptide synthesis, but also enriches the molecular diversity of polypeptides for cosmetics by introducing the unique structure of theanine, and lays a foundation for developing a new skin care ingredient with double activities of theanine and polypeptides.

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:

[0007] 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.

[0008] Preferably, the solid-liquid ratio of the L-theanine and the saturated sodium bicarbonate aqueous solution is 1g: (15-25) mL.

[0009] 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 group or other groups of theanine to subsequent coupling, and solve the core problem that theanine cannot directly participate in solid-phase synthesis. At the same time, 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, which 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, and ensure the purity of the product.

[0010] Preferably, the molar ratio of L-theanine and Fmoc-Osu is 1: (1-1.2).

[0011] Preferably, the specific conditions of the divided addition are: divided into 3 times, and each time interval is 8-12 min.

[0012] Preferably, the specific conditions of the reaction are: the temperature is 20-25℃, the reaction time is 20-25h, and the pH value of the system is maintained at 8.0-8.5.

[0013] The specific steps of the purification are: 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 phase, drying, and filtering to obtain the filtrate, concentrating to 1 / 10 of the volume of saturated sodium bicarbonate aqueous solution, and then loading a silica gel chromatography column for purification.

[0014] Preferably, the mass fraction of citric acid in the citric acid aqueous solution is 10%.

[0015] Preferably, during the extraction, the addition amount of ethyl acetate for each extraction is 70%-80% of the volume of saturated sodium bicarbonate aqueous solution.

[0016] Preferably, the specific steps of the extraction are: adding ethyl acetate to the sample system to be treated, oscillating thoroughly, and then standing and separating. 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.

[0017] 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 within 2-3 mm in diameter on the baseline, if the concentration is low, repeating the spotting 1-2 times 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. The distance from the center of each spot to the baseline and the distance from the front of the developing agent to the baseline are measured, and the Rf value of each spot is calculated.

[0018] Preferably, the specific conditions of the drying are as follows: the drying agent is anhydrous sodium sulfate, the amount of addition is consistent with the mass of L-theanine, and the drying time is 1.5-2.5 h.

[0019] Preferably, the silica gel chromatographic column has a mesh size of 200-300 mesh.

[0020] Preferably, the specific steps of the chromatographic purification are as follows: after the concentrated liquid is loaded, the eluent is used for elution. According to the properties of the target compound, a series of gradient concentrations of methanol-dichloromethane mixed eluent (such as starting from pure dichloromethane, gradually increasing the proportion of methanol, and the mass fraction of methanol being 5%, 10%, 20%··· until pure methanol) is prepared in advance, each concentration of eluent is fully mixed and ultrasonically degassed for 10 minutes, and is placed for standby use.

[0021] Preferably, the flow rate during the elution is 2 mL / min.

[0022] 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.

[0023] The second aspect of the present application provides Fmoc-Tea-OH prepared by the preparation method of Fmoc-Tea-OH, and the structural formula is as follows: Figure 1 .

[0024] The third aspect of the present application provides the application of Fmoc-Tea-OH in polypeptide synthesis, which is applied to the preparation of linear peptides and cyclic peptides.

[0025] The synthesis method of the linear peptide comprises the following steps: taking resin 1 as a carrier, and coupling amino acids from C-terminal to N-terminal according to the amino acid arrangement order of the required linear peptide.

[0026] 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.

[0027] Preferably, the C-terminal amide modification of the resin 1 has a substitution value of 0.5-1.0 mmol / g.

[0028] In some preferred embodiments, the resin 1 is a Rink Amide-AM resin.

[0029] 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.

[0030] Preferably, in the specific steps of the swelling, the ratio of the resin 1 to DMF is 1 mmol:(40-60) mL.

[0031] 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.

[0032] 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.

[0033] Preferably, the deprotection agent is any one of a 50% morpholine-DMF mixed solution or a 20% piperidine-DMF solution.

[0034] Preferably, the ratio of the resin 1 to the deprotection agent is 1 mmol:(40-60) mL.

[0035] The specific steps of washing the resin are as follows: the resin is washed with an organic solvent, and then drained for standby.

[0036] The organic solvent includes one or more of DMF, methanol, dichloromethane, and DMF, and the washing times of each organic solvent are independently 1-3 times.

[0037] Preferably, the sequence of coupling the amino acid is to sequentially couple from the C-terminal to the N-terminal according to the sequence.

[0038] The coupling of the amino acid is a cyclic process, and the coupling methods of all the amino acids are 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.

[0039] 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.

[0040] 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.

[0041] Preferably, the condensing agent 1 includes HBTU, HOBT, DIPEA, and the molar ratio is 1:1:2.

[0042] Preferably, the molar ratio of the Fmoc-protected amino acid and the condensing agent 1 is 1:4.

[0043] 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.

[0044] 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 step 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 captures the by-products that may be generated, inhibiting the racemization side reaction. The addition of double amount of DIPEA provides an alkaline environment, which not only promotes the deprotonation of the carboxyl group, but also neutralizes the protons released during the reaction and the acidic by-products, maintaining a neutral to alkaline environment in the system. In addition, the excess DIPEA can also inhibit the self-cyclization of tea amino acid amino and other side reactions. Through the design of molar ratio, the efficient, stable, and low side reaction coupling of tea amino acid and conventional amino acid is finally achieved.

[0045] Preferably, in the specific steps of the coupling amino acid, the ratio of resin 1 and DMF is 1 mmol:(40-60) mL.

[0046] 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.

[0047] 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, and 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.

[0048] 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.

[0049] Preferably, the cutting solution comprises TFA, Tis and water, and the mass ratio is 95:2.5:2.5.

[0050] Preferably, the ratio of the resin 1 and the cutting solution is 1 mmol:(70-90) mL.

[0051] 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.

[0052] The specific steps of the purification and freeze-drying are as follows: the crude peptide chain is added with ether at -20℃, and after the precipitation for 15 min, 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, and after the reverse phase HPLC purification, the main peak is collected, and the freeze-drying is performed to obtain a white powder.

[0053] Preferably, the ratio of the resin 1 and the ether is 1 mmol:(350-450) mL.

[0054] Preferably, the specific conditions of the centrifugation are as follows: the rotation speed is 4000 rpm, and the time is 10 min.

[0055] Preferably, the specific steps of the reverse HPLC purification are as follows: ① sample pretreatment: dissolve the sample to be purified with the initial mobile phase, and control the concentration at 5-20 mg / mL to ensure complete dissolution of the sample. Filter the sample solution with a 0.22 μm organic phase filter to remove particulate impurities and prevent column blockage. ② Chromatography system preparation: select a reverse phase chromatography column (commonly a C18 column with a specification of 4.6x250 mm and a particle size of 5 μm), prepare the mobile phase, and degas the mobile phase by ultrasonic degassing for 15 minutes and filtering with a 0.22 μm filter. Turn on the HPLC system, set the column temperature to 35°C, connect the chromatography column, and equilibrate the system with the initial mobile phase to the baseline. ③ Chromatography condition setting: flow rate is 1.0 mL / min, detection wavelength is selected according to the ultraviolet absorption characteristics of the target compound (e.g. 254 nm, 210 nm, etc.), and gradient elution program is set according to the 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: use a sample needle to take an appropriate amount of sample solution, inject the sample, start the running program, and record the chromatogram. Observe the peak elution of the chromatographic peak, determine the target peak position according to the retention time, and when the target peak appears, open the fraction collector to collect the corresponding effluent. ⑤ Post-treatment: combine the collected liquids of the same target components, remove the solvent by rotary evaporation to obtain the purified sample, and perform HPLC purity detection on the purified product.

[0056] Preferably, the mobile phase includes 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%.

[0057] Preferably, the organic phase is mobile phase B, and 5% organic phase is a mixed liquid of 5% mobile phase B + 95% mobile phase A.

[0058] Preferably, the specific conditions of the freeze-drying are as follows: temperature is -80°C, and time is 24 h.

[0059] The synthesis method of the cyclic peptide comprises the following steps: using resin 2 as a carrier, first synthesizing a linear peptide containing theanine, and then intramolecular cyclization.

[0060] Preferably, the synthesis method of the cyclic peptide comprises the following steps: after swelling the resin 2, loading the first amino acid, sequentially coupling the amino acids in the direction from the C-terminal to the N-terminal according to the sequence of the cyclic peptide to synthesize a linear peptide chain, then sequentially performing intramolecular cyclization, cutting and purification, and finally freeze-drying to obtain the cyclic peptide.

[0061] Preferably, the substitution value of the resin 2 is 0.3-0.7 mmol / g.

[0062] In some preferred embodiments, the resin 2 is a 2-CTC Resin resin.

[0063] Preferably, the swelling step of the resin 2 is consistent with the specific step of swelling of the resin 1.

[0064] The resin is selected differently according to the structure of the target peptide. For linear peptides, Rink Amide-AM resin is selected. This is to directly obtain C-terminal amidated polypeptides, which more simulate the natural state of polypeptides, and at the same time, there is no need to introduce additional amidation reagents, thus simplifying the synthesis process. For cyclic peptides, linear synthesis is required before cyclization, and 2-CTC Resin resin is selected. This resin has the characteristics of easy cutting and high efficiency of loading amino acids, which is convenient for subsequent cutting, and does not require a side chain protection group, thus simplifying the pretreatment before cyclization.

[0065] The specific steps of loading the first amino acid are similar to the specific steps of coupling the amino acid described above, and specifically, the DMF is drained, the Fmoc-protected amino acid and the condensing agent 2 are dissolved in DMF, and after ultrasonic dissolution, the swollen resin 2 is added. After shaking at 200-250 rpm for 1.5-2.5 h, the resin is washed in sequence to remove unreacted reagents.

[0066] Preferably, the condensing agent 2 is DIPEA.

[0067] Preferably, the molar ratio of the Fmoc-protected amino acid and the condensing agent 2 is 1:1.

[0068] Preferably, the ratio of the total molar amount of the Fmoc-protected amino acid and the condensing agent 2 to the molar amount of the resin 1 is (5-10):1.

[0069] The specific steps of washing the resin are consistent with the steps of washing the resin in the linear peptide synthesis process described above.

[0070] In the synthesis method of the cyclic peptide, the specific steps of amino acid coupling are deprotection first, and then coupling reaction.

[0071] The specific steps of deprotection are the steps of removing the Fmoc protection group and washing the resin in the linear peptide synthesis process described above.

[0072] The specific steps of the coupling reaction are the steps of coupling the amino acid in the linear peptide synthesis process described above, except that the resin 1 in the steps of coupling the amino acid in the linear peptide synthesis process described above is replaced by the resin 2, and at the same time, 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.

[0073] The specific steps of the intramolecular cyclization are N-terminal deprotection first, and then cyclization reaction.

[0074] The specific step of the N-terminal deprotection is the removal of the Fmoc protecting group and the washing of the resin in the linear peptide synthesis process described above.

[0075] The specific step of the cyclization reaction is: adding DMF aqueous solution, then adding condensing agent 3, and then shaking on a 200-250 rpm shaker for 8-12 h.

[0076] Preferably, the mass fraction of DMF in the DMF aqueous solution is 50%.

[0077] Preferably, the ratio of the resin 2 and the DMF aqueous solution is 1 mmol: (80-120) mL.

[0078] The intramolecular cyclization in the DMF aqueous solution effectively suppresses the side reaction of intermolecular polymerization by greatly reducing the concentration of the peptide chain, significantly improves the selectivity and yield of the intramolecular head-to-tail cyclization, and thus successfully synthesizes the tea-containing cyclic peptide with a clear structure, filling the blank of tea-containing cyclic peptide synthesis.

[0079] Preferably, the condensing agent 3 includes HBTU, HOBT, and DIPEA, and the molar ratio is 1:1:2.

[0080] Preferably, the molar ratio of the resin 2 and the condensing agent 3 is 1:12.

[0081] The specific steps of the cyclic peptide cleavage and purification are consistent with the specific steps of the peptide chain cleavage and purification and freeze-drying in the linear peptide synthesis process described above.

[0082] Compared with the prior art, the advantages and beneficial effects of the present application are:

[0083] 1. The present application provides a preparation method of Fmoc-Tea-OH, which uses L-theanine and Fmoc-Osu as raw materials, reacts in a weak alkaline aqueous system, and obtains high-purity Fmoc-Tea-OH through purification. On this basis, by using the Fmoc solid-phase synthesis strategy and optimizing the condensation system, the precise coupling of theanine and other amino acids is successfully realized, and the target polypeptide with a clear structure and up-to-standard purity is obtained.

[0084] 2. The present application solves the problem that theanine is difficult to participate in solid-phase synthesis of polypeptides, not only fills the application blank of theanine in the field of polypeptide synthesis, but also enriches the molecular diversity of polypeptides for cosmetics by introducing the unique structure of theanine, and lays a foundation for developing new skin care ingredients with dual activity of theanine and polypeptides.

[0085] 3. The present application solves the technical bottleneck that theanine is difficult to directly participate in the solid-phase synthesis of polypeptides, provides a synthesis scheme of the adapted theanine derivative Fmoc-Tea-OH, breaks through the compatibility limitation of theanine in the construction of polypeptide sequences, and realizes efficient coupling of theanine and other amino acids.

[0086] 4. The present application solves the technical bottleneck that theanine is difficult to directly participate in the solid-phase synthesis of polypeptides, provides a synthesis scheme of the adapted theanine derivative Fmoc-Tea-OH, breaks through the compatibility limitation of theanine in the construction of polypeptide sequences, and realizes efficient coupling of theanine and other amino acids.

[0087] 5. The present application solves the technical bottleneck that theanine is difficult to directly participate in the solid-phase synthesis of polypeptides, provides a synthesis scheme of the adapted theanine derivative Fmoc-Tea-OH, breaks through the compatibility limitation of theanine in the construction of polypeptide sequences, and realizes efficient coupling of theanine and other amino acids.

[0088] 6. The present application solves the technical bottleneck that theanine is difficult to directly participate in the solid-phase synthesis of polypeptides, provides a synthesis scheme of the adapted theanine derivative Fmoc-Tea-OH, breaks through the compatibility limitation of theanine in the construction of polypeptide sequences, and realizes efficient coupling of theanine and other amino acids.

[0089] 7. The present application solves the technical bottleneck that theanine is difficult to directly participate in the solid-phase synthesis of polypeptides, provides a synthesis scheme of the adapted theanine derivative Fmoc-Tea-OH, breaks through the compatibility limitation of theanine in the construction of polypeptide sequences, and realizes efficient coupling of theanine and other amino acids. BRIEF DESCRIPTION OF DRAWINGS

[0090] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description.

[0091] Figure 1 The structural formula of Fmoc-Tea-OH prepared by the present application;

[0092] Figure 2 The hydrogen spectrum of Fmoc-Tea-OH prepared by the present application;

[0093] Figure 3 The HPLC spectrum of the linear peptide prepared in Example 2 of the present application;

[0094] Figure 4 The HPLC spectrum of the linear peptide prepared in Example 3 of the present application;

[0095] Figure 5 HPLC profile of the linear peptide prepared for the present application Example 4;

[0096] Figure 6 HPLC profile of the linear peptide prepared for the present application Example 5;

[0097] Figure 7 HPLC profile of the linear peptide prepared for the present application Example 6;

[0098] Figure 8 HPLC profile of the linear peptide prepared for the present application Example 7;

[0099] Figure 9 HPLC profile of the cyclic peptide prepared for the present application Example 8;

[0100] Figure 10 HPLC profile of the cyclic peptide prepared for the present application Example 9. DETAILED DESCRIPTION

[0101] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without any creative work, fall within the protection scope of the present application.

[0102] The raw materials used in the present application are all commercially available, and specifically:

[0103] Resin 1 is Rink Amide-AM resin, C-terminal amide-modified, with a substitution value of 0.7 mmol / g, from Macrolin.

[0104] Fmoc-protected amino acids, all from Macrolin.

[0105] Resin 2 is 2-CTC Resin resin, with a substitution value of 0.5 mmol / g, from Macrolin.

[0106] The silica gel chromatographic column has a mesh size of 200-300 mesh, from Macrolin.

[0107] Example 1

[0108] The embodiment provides a Fmoc-Tea-OH, a preparation method thereof, and steps are as follows: 10.0g of L-theanine (54.3mmol) is added with 200ml of saturated sodium bicarbonate aqueous solution, stirred at 300rpm until dissolved (pH is about 8.3), Fmoc-Osu is added in batches under stirring to react, after the reaction is completed, purification is carried out, single component eluent is combined, and white solid crude product is obtained by rotary evaporation, which is Fmoc-Tea-OH.

[0109] The specific conditions of the batch addition are as follows: 3 times in total, and 10min is added each time.

[0110] The specific conditions of the reaction are as follows: the temperature is 23℃, the reaction time is 24h, and the pH value of the system is maintained at 8.0-8.5.

[0111] The specific steps of the purification are as follows: the pH value of the system is adjusted to 3.5 by using 10% citric acid aqueous solution, 150ml of ethyl acetate is used for extraction for 3 times, TLC detection is used for each extraction, until L-theanine spots disappear, the organic phase is combined, dried, filtered, concentrated to 1 / 10 of the volume of saturated sodium bicarbonate aqueous solution, and then loaded on a silica gel chromatography column for purification.

[0112] The specific steps of the extraction are as follows: ethyl acetate is added to a sample system to be treated, oscillated fully, and then static stratification is carried out. After the interface between the two phases is clear, the lower liquid is slowly discharged through a separatory funnel piston, and the upper ethyl acetate extraction liquid is collected and reserved.

[0113] The specific steps of the TLC detection are as follows: a silica gel thin layer plate is lightly drawn with a pencil at a distance of 1.5-2cm from the bottom edge to draw a baseline parallel to the bottom edge, an appropriate amount of sample solution is taken by using a micro-capillary tube, sample spotting is carried out on the baseline, the sample spotting diameter is controlled within 2-3mm, if the concentration is low, 1-2 times of repeated sample spotting can be carried out after the first spot is dried, 5% methanol-dichloromethane developing agent is prepared by taking dichloromethane and methanol in a volume ratio of 95:5, mixing uniformly, and then pouring into a developing cylinder, and the number, position and color of spots are observed and recorded by using a 254nm ultraviolet lamp. The distance between the center of each spot and the baseline and the distance between the front of the developing agent and the baseline are measured, and the Rf value of each spot is calculated.

[0114] 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 2h.

[0115] The specific steps of the chromatographic purification are as follows: after the concentrated liquid is loaded, a series of gradient concentrations of methanol-dichloromethane mixed eluent (starting from pure dichloromethane, gradually increasing the proportion of methanol, and the mass fraction of methanol being 5%, 10%, 20%, and so on until pure methanol) is prepared according to the properties of the target compound, each concentration of eluent is fully mixed and ultrasonically degassed for 10 minutes, and is placed for standby.

[0116] The flow rate during elution is 2 mL / min.

[0117] The specific conditions of the rotary evaporation are as follows: the water bath temperature is 45°C, the vacuum degree is 0.085 MPa, and the rotation speed is 60 rpm.

[0118] The chemical structural formula of the prepared Fmoc-Tea-OH is as follows:

[0119] ;

[0120] The Fmoc-Tea-OH hydrogen spectrum is shown in 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).

[0121] The purity of the prepared Fmoc-Tea-OH is 95.2%, and the yield is 88.9%.

[0122] Example 2

[0123] This embodiment provides a linear peptide containing theanine, and the amino acid sequence is Glu-Glu-Tea-Gln-Arg-Arg-NH2. The synthesis method and steps are as follows: after 0.1 mmol (about 143 mg) of resin 1 is swelled, the C-terminal amino acid is fixed on the swelled resin 1, according to the amino acid arrangement order of the required linear peptide, the Fmoc protecting group is removed in turn, the resin is washed, and the amino acid is coupled. After the synthesis is completed, the peptide chain is cut, and after purification and freeze-drying, the final linear peptide chain is obtained, which is the linear peptide.

[0124] The specific steps of the swelling are as follows: the resin 1 is placed in a T-shaped polypeptide solid-phase synthesis tube, 5 mL of DMF is added, and the shaking table is shaken.

[0125] The specific conditions of the shaking table oscillation are: rotation speed of 280 rpm and time of 15 min.

[0126] The specific steps of removing the Fmoc protecting group are: after the DMF is drawn, 5 mL of the deprotection agent is added, the shaking table is oscillated at 220 rpm for 10 min, then the deprotection agent is drawn and 5 mL of the deprotection agent is added, and oscillation is performed for 20 min.

[0127] The deprotection agent is a piperidine-DMF solution with a mass fraction of 20% of piperidine.

[0128] The specific steps of washing the resin are: the resin is alternately washed with DMF (3 times, 5 mL each time) and DCM (3 times, 5 mL each time), and then drawn and ready for use.

[0129] The order of coupling the amino acids is: sequentially coupling from the C-terminal to the N-terminal according to the sequence, the amino acid sequence of the straight-chain peptide in this embodiment is Glu-Glu-Tea-Gln-Arg-Arg-NH2, the first round of coupling is Arg, and then the second Arg, the third Gln, the fourth Tea (Fmoc-Tea-OH prepared in Example 1), the fifth Glu, and the sixth Glu are sequentially coupled from the C-terminal to the N-terminal according to the sequence.

[0130] The coupling of the amino acids is a cyclic process, the coupling methods of all the amino acids are consistent, and after coupling, the removal of the Fmoc protecting group and the washing of the resin are sequentially performed, and after each coupling, the Kaiser reagent is used for detection, and the resin is colorless.

[0131] Taking the first round of coupling Arg as an example, the specific steps of coupling the amino acids are: 0.4 mmol of Fmoc-Arg (Pbf)-OH and 1.6 mmol of the condensing agent 1 are dissolved in 5 mL of DMF, after ultrasonic dissolution, the swelled resin 1 is added, and then the shaking table is oscillated at 220 rpm for 2 h.

[0132] The condensing agent 1 is HBTU, HOBT, and DIPEA, and the molar ratio is 1:1:2.

[0133] The specific conditions of the ultrasonic dissolution are: ultrasonic time of 2 min, ultrasonic power of 240 W, ultrasonic temperature of 25°C, and ultrasonic frequency of 25 kHz.

[0134] The specific steps of cutting the peptide chain are: after the synthesis is completed, the resin is sequentially washed with DMF (3 times, 5 mL each time) and methanol (3 times, 5 mL each time) and vacuum dried, 8 mL of the cutting solution is added, oscillation is performed at 200 rpm for 2 h, the cutting solution is collected and the resin is washed, the cutting is repeated once, the two cutting solutions are combined, nitrogen blowing is performed until 1 mL, and the crude peptide chain is obtained.

[0135] The specific conditions of the vacuum drying are: vacuum degree of 7.5 Pa, temperature of 25℃, and time of 1 h.

[0136] The cutting liquid comprises TFA, Tis and water, and the mass ratio is 95:2.5:2.5.

[0137] The specific steps of the purification and freeze-drying are: 40 mL of -20℃ ether is added to the crude peptide chain, and the precipitation is centrifuged after 15 min, the supernatant is discarded, and the precipitate is dissolved with 5 mL of trifluoroacetic acid- dichloromethane solution with a mass fraction of 0.1% trifluoroacetic acid. After reverse phase HPLC purification, the main peak is collected, and white powder is obtained by freeze-drying.

[0138] The specific conditions of the centrifugation are: rotation speed of 4000 rpm, and time of 10 min.

[0139] The specific steps of the reverse HPLC purification are: ① sample pretreatment: the sample to be purified is dissolved with the initial mobile phase, and the concentration is controlled at 15 mg / mL to ensure complete dissolution of the sample. The sample solution is filtered with a 0.22 μm organic phase filter to remove particulate impurities and prevent column blockage. ② Chromatography system preparation: select a C18 reverse phase chromatography column with a specification of 4.6×250 mm and a particle size of 5 μm. Prepare the mobile phase, degas the mobile phase for 15 minutes by ultrasonic, and filter with a 0.22 μm filter. Turn on the HPLC system, set the column temperature to 35℃, connect the chromatography column, and equilibrate the system with the initial mobile phase to the baseline. ③ Chromatography condition setting: flow rate of 1.0 mL / min, detection wavelength of 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: use a sample needle to take 30 μL of sample solution, inject the sample, start the running program, record the chromatogram, and observe the peak elution of the chromatographic peak. When the target peak appears, open the fraction collector to collect the corresponding effluent. ⑤ Post-processing: combine the collected liquids of the same target components, remove the solvent by rotary evaporation to obtain the purified sample, and detect the purity of the purified product by HPLC.

[0140] The mobile phase is mobile phase A and mobile phase B, the mobile phase A is TFA aqueous solution with a mass fraction of 0.1% TFA, and the mobile phase B is TFA-acetonitrile solution with a mass fraction of 0.1% TFA.

[0141] The specific conditions of the freeze-drying are: temperature of -80℃, and time of 24 h.

[0142] The amino acid sequence of the prepared linear peptide containing theanine is Glu-Glu-Tea-Gln-Arg-Arg-NH2, and its structural formula is:

[0143] ;

[0144] The HPLC detection result is shown in Figure 3 From the Figure 3 and mass spectrometry results, it can be seen 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).

[0145] Example 3

[0146] This example provides a linear peptide containing theanine, and the amino acid sequence is Gly-Pro-Gln-Tea-Pro-Gln. The synthesis method is the same as that of Example 2, and the difference is that the required Fmoc-protected amino acids are selected according to the amino acid sequence for coupling in turn.

[0147] The prepared linear peptide containing theanine has an amino acid sequence of Gly-Pro-Gln-Tea-Pro-Gln, and its structural formula is:

[0148] ;

[0149] The HPLC detection result is shown in Figure 4 From the Figure 4 and mass spectrometry results, it can be seen 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).

[0150] Example 4

[0151] This example provides a linear peptide containing theanine, and the amino acid sequence is Phe-Val-Tea-Pro-Phe-Pro. The synthesis method is the same as that of Example 2, and the difference is that the required Fmoc-protected amino acids are selected according to the amino acid sequence for coupling in turn.

[0152] The prepared linear peptide containing theanine has an amino acid sequence of Phe-Val-Tea-Pro-Phe-Pro, and its structural formula is:

[0153] ;

[0154] The HPLC detection result is shown in Figure 5 From the Figure 5 and mass spectrometry results, it can be seen 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).

[0155] Example 5

[0156] 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.

[0157] 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:

[0158] ;

[0159] 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).

[0160] Example 6

[0161] 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.

[0162] 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:

[0163] ;

[0164] 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).

[0165] Example 7

[0166] 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.

[0167] The prepared theanine-containing linear peptide has the amino acid sequence Gly-Pro-Arg-Pro-Tea-NH2 and its structural formula is as follows:

[0168] ;

[0169] 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).

[0170] Example 8

[0171] 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.

[0172] The swelling steps of resin 2 are the same as the specific steps of resin 1 swelling in Example 2.

[0173] 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.

[0174] The condensing agent 2 is DIPEA.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] The specific steps of the intramolecular cyclization are as follows: first, N-terminal deprotection is performed, and then a cyclization reaction is carried out.

[0179] The specific step of the N-terminal deprotection is the Fmoc deprotection and resin washing step in the linear peptide synthesis process of Example 2.

[0180] The specific step of the cyclization reaction is: adding 10 mL of DMF aqueous solution, then adding 1.2 mmol of condensing agent 3, and oscillating on a 220 rpm shaker for 10 h.

[0181] The mass fraction of DMF in the DMF aqueous solution is 50%.

[0182] The condensing agent 3 is HBTU, HOBT, and DIPEA, and the molar ratio is 1:1:2.

[0183] The specific steps of the cyclic peptide cleavage and purification are the same as the specific steps of the peptide chain cleavage and purification and freeze-drying in the linear peptide synthesis process of Example 2.

[0184] The prepared cyclic peptide cyclo(Tea-Pro-Gln-Tea-Pro-Gln) containing theanine has the structural formula:

[0185] ;

[0186] The HPLC detection result is shown in Figure 9 . From the Figure 9 and mass spectrometry results, it can be seen 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).

[0187] Example 9

[0188] This example provides a cyclic peptide cyclo(Phe-Val-Tea-Pro-Phe-Pro) containing theanine, and the amino acid sequence is Phe-Val-Tea-Pro-Phe-Pro. The synthesis method is the same as that of Example 8, and the difference is that the required Fmoc-protected amino acid is selected according to the amino acid sequence for coupling.

[0189] The prepared cyclic peptide cyclo(Phe-Val-Tea-Pro-Phe-Pro) containing theanine has the structural formula:

[0190] ;

[0191] The HPLC detection result is shown in Figure 10 . From the Figure 10 and mass spectrometry results, it can be seen 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).

[0192] 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.

[0193] 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 by, The method comprises the following steps: adding saturated sodium bicarbonate solution to L-theanine, stirring at 200-400 rpm until dissolved, adding Fmoc-Osu in portions under stirring to react, purifying after the reaction, combining single component eluent, and rotary evaporating to obtain white solid crude product, which is Fmoc-Tea-OH; the solid-liquid ratio of the L-theanine and the saturated sodium bicarbonate solution is 1 g:(15-25) mL; the molar ratio of the L-theanine and Fmoc-Osu is 1:(1-1.2); 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; 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, filtering to obtain filtrate, concentrating to 1 / 10 of the volume of saturated sodium bicarbonate aqueous solution, and then purifying by silica gel column chromatography; the structural formula of the Fmoc-Tea-OH is as follows: 。

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