A polypeptide, and a preparation method and application thereof

Abstract

The present application relates to the technical field of polypeptide, in particular to a polypeptide and a preparation method and application thereof.The polypeptide of the present application is shown as formula (I) R1-HX1X2HPLSPS-OH.The polypeptide of the present application can be added to a drug, food or cosmetic as a surfactant, and has good foaming capacity and anti-cold turbidity capacity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polypeptides, in particular to a polypeptide and a preparation method and application thereof. BACKGROUND

[0002] A polypeptide is a bioactive molecule formed by two or more amino acids connected by a peptide bond, and its molecular weight is usually between that of a small molecule compound and a protein. Compared with proteins, polypeptides have the significant characteristics of clear molecular structure, high controllability of synthesis, and good biocompatibility, and the programmability of the amino acid sequence in the molecular chain provides a broad space for functional design.

[0003] At present, polypeptides have been industrialized and applied in multiple fields: in the field of biological medicine, they are used as drug carriers, vaccine adjuvants, and active ingredients combined with target points; in the cosmetics industry, they are usually used as active raw material ingredients, such as the commonly seen tripeptide-1 and acetyl hexapeptide-8; in food processing, they are used as functional additives and excipients; and in the agricultural field, they are used as biological stimulants. Some polypeptides have certain surface activity due to the combination of hydrophobic groups and hydrophilic groups, and thus can also be used in the field of surfactants. For example, the patent application with the Chinese patent publication number CN110872357A discloses a polypeptide surfactant for oil displacement and demulsification. For another example, the patent application with the Japanese patent publication number JP1993255386A discloses a polypeptide surfactant that can be used for foaming and stain removal. Polypeptide surfactants have the characteristics of high biological safety and strong surface activity, and thus have gradually become one of the hotspots in the development of surfactants. SUMMARY

[0004] The purpose of the present application is to provide a polypeptide with surface activity, which has the characteristics of acid and alcohol resistance and can be widely used in the fields of medicines, foods, and cosmetics.

[0005] In view of this, the present application provides a peptide represented by formula (I), or a stereoisomer thereof or a mixture of stereoisomers,

[0006] R1-HX1X2HPLSPS-OH (I)

[0007] In formula (I),

[0008] X1 and X2 are each selected from one of -Gln-, -Glu-, -Asn-, and -Asp-;

[0009] R1- is selected from NH2-PEGn-CO-, and n is selected from 2-4.

[0010] PEG refers to polyethylene glycol polymerized from ethylene glycol monomers, and n in PEGn refers to the degree of polymerization of the PEG. For example, PEG2 refers to a polyethylene glycol polymerized from two ethylene glycol monomers. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4.

[0011] X1and X2each refer to an amino acid residue, and the selection of X1and X2is independent of each other. For example, when X1is -Gln-, X2may also be -Gln-. In some embodiments, X1and X2differ by only one group or are identical, for example, when X1is -Gln-, X2is -Gln- or -Glu-; when X1is -Asn-, X2is -Asn- or -Asp-. In some embodiments, X1and X2are independently selected from -Gln- and -Glu-. In some embodiments, X1and X2are independently selected from -Asn- and -Asp-.

[0012] In some embodiments, the peptide of formula (I), or a stereoisomer, or a mixture of stereoisomers thereof, is selected from the following peptides (1)-(8):

[0013] (1) NH2-PEG2-CO-HQQHPLSPS-OH;

[0014] (2) NH2-PEG2-CO-HQEHPLSPS-OH;

[0015] (3) NH2-PEG2-CO-HEQHPLSPS-OH;

[0016] (4) NH2-PEG2-CO-HDDHPLSPS-OH;

[0017] (5) NH2-PEG2-CO-HDNHPLSPS-OH;

[0018] (6) NH2-PEG2-CO-HNDHPLSPS-OH;

[0019] (7) NH2-PEG2-CO-HNNHPLSPS-OH;

[0020] (8) NH2-PEG2-CO-HEEHPLSPS-OH.

[0021] In some embodiments, the peptide of formula (I), or a stereoisomer, or a mixture of stereoisomers thereof, is selected from the following peptide (4).

[0022] The peptides of formula (I) of the present invention may exist as stereoisomers or mixtures of stereoisomers; for example, the amino acids they contain may have L-, D-, or independently racemic configurations. Thus, it is possible to obtain isomeric mixtures, as well as racemic or diastereomeric mixtures, or pure diastereomers or enantiomers, depending on the number of asymmetric carbon atoms and the presence of isomers or isomeric mixtures. Preferred structures of the peptides of formula (I) of the present invention are pure isomers, i.e., enantiomers or diastereomers. The naturally occurring L-isomer may be preferred.

[0023] The present invention also encompasses all suitable isotopic variants of the peptides represented by formula (I). Isotopic variants of these peptides of the present invention are understood herein to mean compounds in which at least one atom within the peptides of the present invention is replaced by another atom of the same atomic number, but having an atomic mass different from the atomic mass usually or predominantly found in nature. Examples of isotopes that can be incorporated into the peptides of the present invention are those of hydrogen, carbon, nitrogen, or oxygen, such as 2H (deuterium), 3H (tritium), 13C, 14C, 14N, 15N, 17O, or 18O. Certain isotopic variants of the compounds of the present invention (particularly those into which one or more radioactive isotopes have been incorporated) may be advantageous, for example, for examining the mechanism of action or distribution of the active compound in vivo; peptides labeled with 3H or 14C are particularly suitable for this purpose due to their relatively simple preparation and detectability. In addition, due to the greater metabolic stability of the peptide, the incorporation of isotopes (e.g., deuterium) can produce specific therapeutic benefits, such as an extension of the half-life in vivo or a reduction in the required active dose; therefore, in some cases, such modifications of the peptides of the present invention may also constitute a preferred embodiment of the present invention. Isotopic variants of the peptides of the present invention can be prepared by methods known to those skilled in the art, for example, by the methods further described below and in the examples, by using the respective reagents and / or corresponding isotopic modifications of the starting materials.

[0024] The term "salt" includes metal salts of the peptide represented by formula (I), wherein the metal includes, but is not limited to, lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc or aluminum; includes salts formed between the peptide represented by formula (I) and an organic base, wherein the organic base includes, but is not limited to, ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine or piperazine; includes salts formed between the peptide represented by formula (I) and an inorganic acid or an organic acid, wherein the organic acid includes, but is not limited to, acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, pamoate or gluconic acid; and the inorganic acid includes, but is not limited to, hydrochloric acid, sulfuric acid, boric acid or carbonic acid.

[0025] The synthesis of the peptide of formula (I) or of its salts can be carried out according to the conventional methods known in the prior art, such as solid-phase synthesis, liquid-phase synthesis or a combination of solid-phase and liquid-phase methods, but also by biotechnological methods aimed at producing the desired sequence, or by controlled hydrolysis of proteins of animal, fungal or plant origin.

[0026] To this end, the application provides a process for obtaining a peptide of formula (I) comprising the following steps:

[0027] Step 1, coupling of an amino acid having a protected N-terminal end and a free C-terminal end with an amino acid having a free N-terminal end and a C-terminal end bound to a solid support;

[0028] Step 2, elimination of the group protecting the N-terminal end;

[0029] Step 3, repeating said step 1 and said step 2 until the desired peptide sequence is obtained;

[0030] Step 4, cleavage of the peptide from the solid support;

[0031] Step 5, coupling of the R1 group with the free N-terminal end of the peptide.

[0032] Preferably, the C-terminal end is bound to a solid support and the process is carried out on solid phase, comprising coupling of an amino acid having a protected N-terminal end and a free C-terminal end with an amino acid having a free N-terminal end and a C-terminal end bound to a polymeric support; elimination of the group protecting the N-terminal end; and repeating this sequence the required number of times in order to thus obtain a peptide having the desired length, followed by cleavage of the synthesized peptide from the initial polymeric support.

[0033] The functional groups of the side chains of these amino acids are kept fully protected throughout the synthesis with temporary or permanent protecting groups, and can be deprotected simultaneously or orthogonally to the process of cleaving the peptide from the polymeric support.

[0034] Alternatively, solid-phase synthesis can be carried out by a convergent strategy by coupling dipeptides or tripeptides to the polymeric support or to a dipeptide or amino acid previously bound to the polymeric support.

[0035] N- and C-terminal deprotection and / or cleavage of the compound from the polymer support in an undetermined order using standard conditions and methods known in the art, followed by modification of the functional groups of the termini can be performed. Optional modification of the N- and C-termini of the compound bound to the polymer support, or after the compound has been cleaved from the polymer support, can be performed.

[0036] In another aspect of the present application, there is provided a use of the peptide represented by the above formula (I) or a stereoisomer thereof, or a mixture of stereoisomers, as a surfactant.

[0037] The peptide of the present application has a surface activity and is effective in maintaining the amount of foam and the duration of the existence of foam in a liquid preparation, and functions as a foaming agent.

[0038] In another aspect of the present application, there is provided a use of the peptide represented by the above formula (I) or a stereoisomer thereof, or a mixture of stereoisomers, in the manufacture of a pharmaceutical product, a food product, or a cosmetic product.

[0039] In another aspect of the present application, there is provided a use of the peptide represented by the above formula (I) or a stereoisomer thereof, or a mixture of stereoisomers, in the manufacture of a liquid composition.

[0040] In some embodiments, the peptide is used to improve the physical stability of the liquid composition.

[0041] In the present specification, the abbreviations used for amino acids follow the rules specified by the IUPAC-IUB Commission of Biochemical Nomenclature in Eur. J. Biochem. 1984, 138: 9-37.

[0042] Thus, for example, Gly represents NH2-CH2-COOH, Gly- represents NH2-CH2-CO-, -Gly represents -NH-CH2-COOH, and -Gly- represents -NH-CH2-CO-. The hyphen representing a peptide bond thus eliminates the OH in the 1-carboxyl group of the amino acid on the right of the symbol (here represented in the conventional non-ionized form) and the H in the 2-amino group of the amino acid on the left of the symbol; both modifications can apply to the same symbol.

[0043] The abbreviations His and H both represent histidine; Gln and Q both represent glutamine; Pro and P both represent proline; Leu and L both represent leucine; Ser and S both represent serine; Glu and E both represent glutamic acid; Asp and D both represent aspartic acid; Asn and N both represent asparagine.

[0044] The present application has the following advantages and effects:

[0045] 1. The peptide of the present application has surface activity, and specifically belongs to a foaming agent, and can be widely applied in the fields of medicines, foods and cosmetics according to the requirements of preparations.

[0046] 2. The peptide of the present application can maintain high stability in an acidic environment and in alcohol-containing preparations, thereby widening the application range of surface active agents.

[0047] 3. The peptide of the present application can maintain high stability after being placed at 80°C for a period of time, and can be applied to medicines, foods and cosmetic products which need to be disinfected by using the pasteurization method.

[0048] 4. The peptide of the present application can effectively inhibit the chill haze phenomenon of beer products, thereby improving the physical stability of beer products. DETAILED DESCRIPTION

[0049] In order to make the objectives, characteristics and advantages of the present application more apparent and easy to understand, the present application will be further described in detail below in combination with examples. Obviously, the described examples are only a part of the examples of the present application, rather than all the examples. Based on the examples in the present application, all other examples obtained by those skilled in the art without making creative efforts fall within the protection scope of the present application.

[0050] Unless otherwise specified, the experimental reagents and materials used in the present application can be obtained by marketing. The following are the abbreviations of some reagents and materials:

[0051] Wang Resin: a starting resin for polypeptide synthesis; Fmoc-Linker: 4-[(2,4-dimethoxyphenyl)(Fmoc-amino)methyl]phenoxyacetic acid; HOBt: 1-hydroxybenzotriazole; DMF: N,N-dimethylformamide; DIC: diisopropyl carbodiimide; DMAP: 4-dimethylaminopyridine; Ac2O: acetic anhydride; DIPEA: diisopropylethylamine; piperidine: piperidine; tBu: tert-butyl; Trt: trityl; TFA: trifluoroacetic acid; TIS: triisopropylsilane; OtBu: tert-butoxy.

[0052] Example 1 Preparation of NH2-PEG2-CO-His-Gln-Gln-His-Pro-Leu-Ser-Pro-Ser-OH

[0053] Step 1, weigh the Wang Resin in the solid-phase synthesis reaction column, swell with DMF, wash the resin, and remove the solvent.

[0054] Step 2, weigh Fmoc-Ser(tBu)-OH and HOBt into a dry flask, dissolve in DMF, cool in ice water bath, activate with DIC, avoid water vapor.

[0055] Step 3, add activated Fmoc-Ser(tBu)-OH and DMAP to the swelled resin, react, wash the resin, remove the solvent; continue to add Ac2O, DIPEA and DMAP to cap. Wash the resin, remove the solvent.

[0056] Step 4, Fmoc-Ser(tBu)-Wang Resin is deprotected with 20% piperidine / DMF, sample K test, deep blue color. Wash the resin with DMF, remove the solvent.

[0057] Step 5, weigh Fmoc-Pro-OH and HOBt into a dry flask, dissolve in DMF, seal in a -18°C environment. Activate with DIC, avoid water vapor. Add the activated amino acid to the deprotected resin, react, remove the reaction solution. K test the resin is colorless and transparent, indicating that the reaction is complete.

[0058] Step 6, the N-terminal Fmoc group is deprotected, and in the presence of HOBt and DIC, using DMF as the solvent, the activated Fmoc-Ser(tBu)-OH is coupled to the peptide-based resin, and the reaction is continued. Then wash the resin and repeat the deprotection of the Fmoc group to couple the next amino acid. In the presence of HOBt and DIC, using DMF as the solvent, sequentially couple Fmoc-Pro-OH, Fmoc-His(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-His(Trt)-OH; after the reaction is complete, wash the resin, remove the solvent.

[0059] Step 7, the N-terminal Fmoc group of the peptide-based resin is deprotected, deprotected with 20% piperidine / DMF, sample K test, deep blue color. Wash the resin with DMF, remove the solvent.

[0060] Step 8, in the presence of HOBt and DIC, using DMF as the solvent, NH2-PEG2-COOH is coupled to the peptide-based resin, the reaction is continued, the resin is washed, the solvent is removed, and after shrink drying, NH2-PEG2-CO-His(Trt)-Gln(Trt)-Gln(Trt)-His(Trt)-Pro-Leu-Ser(tBu)-Pro-Ser(tBu)-Wang Resin is obtained.

[0061] Step 9, TFA, TIS and water were mixed in a volume ratio of 38.5:1:1 to obtain a lysis solution, which was sealed and stored in a refrigerator at -18°C for standby; isopropyl ether was stored in a refrigerator at -18°C for standby.

[0062] Step 10, NH2-PEG2-CO-His(Trt)-Gln(Trt)-Gln(Trt)-His(Trt)-Pro-Leu-Ser(tBu)-Pro-Ser(tBu)-Wang Resin was weighed into a round-bottom flask, and the above-mentioned frozen lysis solution was added, and the reaction was stirred. The filtrate was collected after filtration, and isopropyl ether was added, stirred, centrifuged, washed, and vacuum dried to obtain NH2-PEG2-CO-His-Gln-Gln-His-Pro-Leu-Ser-Pro-Ser-OH crude peptide.

[0063] Step 11, the crude peptide was dissolved in acetic acid and ultrasonically dissolved until no obvious particles were visually observed. Methanol and pure water were added, and the clear transparent solution was obtained by microfiltration. The filtered sample was injected for purification by reverse phase HPLC, and the fraction was collected, concentrated, and freeze-dried to obtain peptide (1) NH2-PEG2-CO-His-Gln-Gln-His-Pro-Leu-Ser-Pro-Ser-OH.

[0064] Peptides (2) to (8) and other polypeptides can be prepared by similar methods. For example, when Glu is needed, Fmoc-Glu(OtBu)-OH is added for coupling reaction; when Asp is needed, Fmoc-Asp(OtBu)-OH is added for coupling reaction; and when Asn is needed, Fmoc-Asn(Trt)-OH is added for coupling reaction.

[0065] Example 2 High-temperature stability test

[0066] Accurately weigh the polypeptide powder, and configure the polypeptide into a 1000 ppm polypeptide solution with water as the solvent. Take a sample, and determine the content by HPLC, which is recorded as t0. Then, seal and place it at 80°C for one hour, take a sample, and determine the content by HPLC, which is recorded as t1. Repeat the above steps three times, and calculate the relative content change rate of the polypeptide. The results are shown in Table 1.

[0067] Relative content change rate of polypeptide = × 100%.

[0068] Relative content change rate of polypeptide = × 100%.

[0069] Table 1 Relative content change rate of polypeptide

[0070]

[0071] From the results, it can be seen that the NH2-PEG2-CO-modification at the N-terminus significantly improves the short-term high-temperature resistance of the polypeptide, while the conventional Ac modification cannot improve the short-term high-temperature resistance of the polypeptide, and the polypeptide without modification at the N-terminus also cannot improve its short-term high-temperature resistance. Improving the short-term high-temperature resistance of the polypeptide is conducive to adding the polypeptide as an additive to products that need to be subjected to the pasteurization method, so that the polypeptide can still maintain a high content level after being subjected to the pasteurization method.

[0072] Example 3 Accelerated test

[0073] Accurately weigh the polypeptide powder, and configure the polypeptide into a 1000 ppm polypeptide solution with 5% ethanol solution as the solvent. Adjust the pH value of the solution to about 4.2 with HCl, take a sample, and determine the content by HPLC, which is recorded as x0. Then seal and place at 37°C for 30 days. Take a sample, determine the content by HPLC, which is recorded as x1. Perform three groups in parallel, and calculate the relative content change rate of the polypeptide. The results are shown in Table 2.

[0074] Relative content change rate of polypeptide = × 100%.

[0075] Table 2 Relative content change rate of polypeptide

[0076]

[0077] From the results, it can be seen that the polypeptide modified by NH2-PEG2-CO- at the N-terminus can be stored for a long time in an acidic and alcohol-containing environment, and its content has hardly changed significantly. The polypeptide modified by the conventional Ac or without modification cannot resist acid and alcohol. The polypeptide that resists acid and alcohol has a wider application space, for example, it can be added as an additive to beer with weak acidity and a certain concentration of ethanol.

[0078] Example 4 Foaming power test

[0079] The foaming power of the peptides (1)-(8) was determined by the Ross-Miles method in GB / T 7462-94. The test sample was taken, and wheat beer (self-brewed) was used as the solvent to configure polypeptide solutions with a mass ratio of 0.01%, 0.1%, 0.2%, and 0.5%, respectively. At 25°C, the foaming volume and foam half-life were recorded according to the operation of the Ross-Miles method. The results are shown in Table 3.

[0080] Table 3 Foam volume of different samples

[0081]

[0082] The results show that, compared with the wheat beer, the polypeptide solution added with the peptides (1)-(8) of the application all have better foaming volume and longer half-life of foam, proving that the peptides (1)-(8) of the application all have the foaming effect and can be used as surfactants and foaming agents. In particular, the addition of the polypeptides of the application in the beer can make the beer have more and longer-lasting foam, which is beneficial to improve the flavor of the beer.

[0083] Example 5: Cooling haze test

[0084] The test sample was used to prepare a polypeptide solution with a mass ratio of 0.5% using wheat beer (self-brewed) as a solvent, and wheat beer (self-brewed) without the addition of polypeptides was used as a negative control. The solution samples were sealed and all placed in a 4°C environment, and after 14 days of standing, the samples were taken and the turbidity of each sample was measured using a turb 555 turbidimeter. The results are shown in Table 4.

[0085] Table 4: Results of cooling haze test

[0086]

[0087] During the cold storage of beer, irreversible haze will occur, which is caused by the precipitation of polyphenol-protein complexes. The change in the turbidity of beer during cold storage can be measured by a turbidimeter. As can be seen from the results in Table 4, the peptides (1)-(8) can effectively inhibit the generation of polyphenol-protein complexes during the cold storage of beer, thereby effectively reducing the degree of cooling haze of beer.

[0088] Although the preferred embodiments of the embodiments of the application have been described, those skilled in the art, once they know the basic creative concept, can make additional changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the embodiments of the application.

[0089] Finally, it should also be noted that, in this document, relational terms such as first and second and the like can only be used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations. Moreover, the terms "comprising", "including" or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, method, article or terminal device including a series of elements not only includes those elements, but also includes other elements not explicitly listed or inherent to such a process, method, article or terminal device. Without more limitations, the element defined by the statement "comprising a" does not exclude the presence of additional identical elements in the process, method, article or terminal device including the element.

[0090] The above has carried on the detailed introduction to the polypeptide, the preparation method and the application provided by the application, the principle and the implementation mode of the application have been described in the text by applying specific examples; the above example explanation is only for helping understanding the method and the core thought of the application; at the same time, for the general technical personnel in the prior art, according to the thought of the application, the specific implementation mode and the application range will have the change, and the above-mentioned, the content of the specification should not be understood as the limitation of the application.

Claims

1. A peptide, characterized in that The structure of the peptide is as follows: NH2-PEG2-CO-HDDHPLSPS-OH.

2. A method for preparing the peptide according to claim 1, comprising the following steps: Step 1, coupling an amino acid having a protected N-terminus and a free C-terminus with an amino acid having a free N-terminus and a C-terminus bound to a solid support; Step 2, eliminating the group protecting the N-terminus; Step 3, repeating steps 1 and 2 until the desired peptide sequence is obtained; Step 4, cleaving the peptide from the solid support; Step 5: Couple NH2-PEG2-COOH to the free N-terminus of the peptide.

3. Use of the peptide according to claim 1 or the peptide prepared by the preparation method according to claim 2 for increasing the foaming performance of beer or reducing the turbidity of cooled beer.

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