阅读说明：本技术 一种硫烯醚化合物及其合成方法和在多肽环化中的应用 (Thioether compound, synthesis method thereof and application thereof in polypeptide cyclization ) 是由 吴川六 李卓儒 郑晓莉 于 2019-10-25 设计创作，主要内容包括：一种硫烯醚化合物及其合成方法和在多肽环化中的应用,属于生物化学技术领域,利用该硫烯醚化合物,可以在温和的条件下对含有一个氮端半胱氨酸及一个链内半胱氨酸的多肽进行快速地环化,从而构建环肽。利用该方法进行翻译后修饰可以构建展示环化多肽的噬菌体展示库,并在此基础上进一步构建可用于检测和治疗等应用的环肽。(A thioether compound, a synthetic method thereof and application thereof in polypeptide cyclization belong to the technical field of biochemistry, and the thioether compound can be utilized to rapidly cyclize a polypeptide containing a nitrogen-terminal cysteine and an intrachain cysteine under mild conditions so as to construct cyclic peptide. The method is utilized to carry out posttranslational modification to construct a phage display library for displaying cyclized polypeptides, and further construct cyclic peptides for detection, treatment and other applications on the basis.)

1. A thioether compound, characterized by the following general structural formula:

wherein n is 2-5, and X is chlorine or bromine.

2. A synthetic method for preparing a thioether compound according to claim 1, comprising the steps of:

1) condensing p-chloroformyl methyl benzoate, m-chloroformyl methyl benzoate or o-chloroformyl methyl benzoate serving as raw materials with malononitrile under the action of sodium hydride to form enol;

2) carrying out hydrolysis reaction under the action of sodium hydroxide to obtain a carboxylic acid intermediate;

3) under the action of phosphorus pentachloride, carrying out chlorination reaction to form chlorinated olefin;

4) under the action of sodium bicarbonate, substituting ethanethiol to obtain a thioether compound;

5) activating by using carbodiimide and N-hydroxysuccinimide to obtain an active ester intermediate;

6) condensing the active ester intermediate with amide obtained by the reaction of straight-chain alkane diamine and halogenated acetyl halide, and substituting with N-acetylcysteine under the action of sodium bicarbonate to obtain a target product;

the synthetic route is as follows:

3. use of a thioether ether compound according to claim 1, for cyclisation of a polypeptide, wherein: the thioether compounds cyclize polypeptides containing one nitrogen-terminal cysteine and one in-chain cysteine to construct cyclic peptides.

4. The use of a thioether ether compound according to claim 3, wherein: adding the polypeptide, a thioether compound, N-acetyl-L-cysteine and tri (2-carboxyethyl) phosphine into a phosphate buffer solution for reaction, thereby cyclizing the polypeptide.

5. The use of a thioether ether compound according to claim 4, wherein: the molar ratio of the polypeptide, the thioether compound, the N-acetyl-L-cysteine and the tri (2-carboxyethyl) phosphine is 1 (1-10) to 1-20; the reaction temperature is 20-40 ℃, and the reaction time is 60-720 min; the pH of the phosphate buffer is 6.0-9.0.

6. The use of a thioether ether compound according to claim 5, wherein: the molar ratio of the polypeptide to the thioether compound to the N-acetyl-L-cysteine to the tris (2-carboxyethyl) phosphine is 1:2:4: 4; the reaction time is 60-120 min; the pH of the phosphate buffer was 7.4.

7. The use of a thioether ether compound according to claim 3, wherein: the nitrogen end of the polypeptide is amino.

8. The use of a thioether ether compound according to claim 7, wherein: the polypeptide is represented by C (Xaa) ac (Xaa) b, C is cysteine, Xaa is selected from the amino acids glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, asparagine, glutamic acid, lysine, glutamine, methionine, serine, threonine, proline, histidine, arginine; a is from 1 to 30, b is from 0 to 29, and the sum of a and b is 30.

9. The use of a thioether ether compound according to claim 4, wherein: the polypeptide displayed on the surface of the phage is subjected to posttranslational modification in a phosphate buffer solution by using a cyclization method based on the thioether compound, so that a phage library displaying cyclic peptide is constructed.

10. The use of a thioether ether compound according to claim 9, for cyclizing a polypeptide, wherein: dropwise adding a phage library of a nitrogen-terminal displayed polypeptide into a phosphate buffer solution, adding tris (2-carboxyethyl) phosphine, incubating for 15-60 min at 20-40 ℃, then adding N-acetyl-L-cysteine and the thioether compound, uniformly mixing, incubating for 60-720 min at 20-40 ℃, then adding an ice PEG/NaCl aqueous solution to precipitate phage, placing in ice for a period of time, finally obtaining phage precipitate through centrifugation, and re-suspending with 1 x phosphate buffer solution to obtain a cyclized phage library.

Technical Field

The invention belongs to the technical field of biochemistry, and particularly relates to a thioether compound, a synthetic method thereof and application thereof in polypeptide cyclization.

Background

In recent years, polypeptides have been widely used in the fields of chemistry, life sciences, and medicine. The polypeptide is a molecule between biological macromolecules and organic micromolecules, and has the advantages of the biological macromolecules and the organic micromolecules. The polypeptide molecule has the characteristics of high diversity, high affinity and high selectivity of biological macromolecules. Compared with the traditional biological macromolecule, the organic macromolecule has the characteristics of easy synthesis, easy derivation and modification of small organic molecules (see European journal of biological chemistry 2015,94,459, Bioorganic & biological chemistry 2018,26, 2759).

Polypeptides in general and linear peptides in particular, exhibit relative flexibility and multiple conformations due to the lack of interactions between peptide chains in similar proteins, which greatly reduces their affinity and selectivity. The loose structure also causes some enzyme cutting sites and reaction sites to be exposed, so that the metabolic stability of the enzyme cutting sites and the reaction sites is problematic. The above problems are solved to some extent by cyclising a generally linear polypeptide to form a cyclic peptide by some cyclisation method. Cyclization limits the conformational flexibility of the polypeptide to a subset of the conformation possessed by the non-cyclized form, such that the polypeptide can better interact with the target by effectively pre-organizing it. By this pre-organization, the entropy of the polypeptide upon binding to the target can be reduced. In some cases, affinity and selectivity may also be improved due to reduced conformation of the polypeptide bound outside the target by cyclization. In addition, the cyclized polypeptide generally exhibits greater rigidity and is more structurally compact, which further increases the metabolic stability and membrane permeability of the polypeptide (see ACS chemical biology2012,7,817, angelwald chemie 2018,57,14414, ACS combinatorial science 2015,17,190, macromolecules 2018, 23).

The most common cyclization method at present is cyclization of polypeptides by disulfide bonds, which is also the predominant form of polypeptide cyclization in organisms. Disulfide bonds are formed by the oxidation of sulfhydryl groups of two cysteine residues on a polypeptide or protein. However, in vitro synthesis, disulfide bonds are susceptible to incorrect pairing, forming incorrect conformations or multimers, due to the lack of a suitable oxidizing environment. In addition, disulfide bonds are less stable than other covalent bonds and are subject to cleavage when subjected to a reducing environment. The above two points make the disulfide bond-based cyclization method unsuitable for practical use. A series of methods have been developed to improve the defect of disulfide bond cyclization. Cyclization by replacing the disulfide bond with a thioether bond is a method which has been widely used. The stability of thioether bond is good, and the defect of unstable disulfide bond is overcome. Whereas the formation of thioether bonds is based on the reaction of substitution of thiol and halogen, which makes the thioether bond method directly applicable to disulfide-bond applicable systems. However, thioether bond-based cyclization methods generally employ highly reactive halogens or halogen-like compounds such as bromine, iodine, and the like. These halogens are liable to undergo side reactions such as hydrolysis and elimination during the reaction. In addition, other groups with higher activity in the polypeptide, such as amino groups, hydroxyl groups, etc., can also react with halogen to produce byproducts. Suga et al solved the problem of side reactions by using low reactive chlorine (Organic & biololecular chemistry2012,10,5783). However, the use process of the catalyst requires the insertion of chlorine-containing unnatural amino acids to meet the reaction conditions of intramolecular reactions, which limits the application range. In addition to the method of cyclization using a thiol group of cysteine, there is a method of utilizing an amino acid residue such as lysine, arginine or tryptophan, and among these methods, the amidation method based on lysine is a relatively common method, but the succinimide active ester used in this method is unstable and is liable to cause side reactions, resulting in a low yield. In addition, methods for unnatural amino acids, such as insertion of an olefin-containing unnatural amino acid and cyclization using olefin metathesis reactions, are also advantageous. An unnatural amino acid containing azide and an alkyne is inserted and cyclized by azide-alkyne cycloaddition. Such methods all require the introduction of unnatural amino acids, limiting the scope of application. And a metal catalyst is usually needed in the reaction, and the metal catalyst may introduce the problem of metal residue and may also cause the denaturation of the polypeptide, thereby further restricting the application of the method (the polypeptide cyclization method is shown in Chemical reviews 2014,114,901).

In addition to modification or cyclization of existing polypeptide sequences, the combination of cyclization method and directed evolution technology of biomolecules is becoming a new trend of research and application in recent years. Compared with the reasonable design of low throughput, the directed evolution technology is a high-throughput method for designing the polypeptide meeting the requirement from the existing structure. Random mutation and recombination are utilized to construct an extremely rich library, selection pressure is given according to specific requirements and purposes, biomolecules with expected characteristics are screened out, and simulated evolution at a molecular level is realized repeatedly. Currently, directed evolution technology has greatly facilitated the development of many fields such as enzyme engineering, metabolic engineering, and medicine (see J Drug Target,2017,25(3),216, Chem Rev,1997,97(2), 391). Henis et al cyclize phage libraries using bromide to construct phage libraries displaying cyclic peptides with abundant structure, and screen serum-released kinases based on the libraries to obtain inhibitor cyclic peptides with high affinity (see nat. chem. biol.,2009,5(7), 502). In addition to phage surface display techniques, directed evolution techniques include the following classes: SICLOPPS, bacterial surface display technology, yeast cell surface display technology, mRNA display technology, mammalian cell surface display technology, and the like. The above directed evolution techniques all need to be performed in an environment close to physiological conditions, which requires that the cyclization method has good biocompatibility. This further requires that the cyclization process must be efficient and selective due to the very low concentration of the reaction system. These requirements make many of the cyclization methods commonly used in chemical synthesis unsuitable for use in directed evolution techniques.

In conclusion, there is still a need in the art to develop more mild and efficient methods for polypeptide cyclization, especially cyclization methods with good biocompatibility, so as to further develop more applications such as polypeptide-based detection and treatment.

Disclosure of Invention

The invention aims to solve the technical problem of complex cyclic peptide synthesis in the prior art, and provides a thioether compound, a synthesis method thereof and application thereof in polypeptide cyclization.

In order to achieve the purpose, the invention adopts the following technical scheme:

a thioether compound, having the following structural formula:

wherein n is 2-5, more preferably n is 2, and X is chlorine or bromine.

A synthetic method for preparing the thioether compound, comprising the steps of:

1) condensing p-chloroformyl methyl benzoate, m-chloroformyl methyl benzoate or o-chloroformyl methyl benzoate serving as raw materials with malononitrile under the action of sodium hydride to form enol;

2) carrying out hydrolysis reaction under the action of sodium hydroxide to obtain a carboxylic acid intermediate;

3) under the action of phosphorus pentachloride, carrying out chlorination reaction to form chlorinated olefin;

4) under the action of sodium bicarbonate, substituting ethanethiol to obtain a thioether compound;

5) activating by using carbodiimide and N-hydroxysuccinimide to obtain an active ester intermediate;

6) condensing the active ester intermediate with amide obtained by the reaction of straight-chain alkane diamine and halogenated acetyl halide, and substituting with N-acetylcysteine under the action of sodium bicarbonate to obtain a target product;

the synthetic route is as follows:

the synthetic steps of the thioether compound are as follows:

(1) sodium hydride was dispersed in tetrahydrofuran and stirred vigorously in an ice bath at 0 ℃ under nitrogen. The malononitrile is dissolved in tetrahydrofuran and slowly added dropwise to the above system. The reaction system is kept in an ice bath at 0 ℃, and after the dropwise addition, the reaction is stirred in the ice bath for 1 h. The compound of formula (1) dissolved in tetrahydrofuran was slowly added dropwise to the system and the reaction system was maintained at 0 ℃. After the completion of the dropwise addition, the reaction system was transferred to room temperature and stirred at room temperature for 1 hour. Tetrahydrofuran in the system was removed by distillation under reduced pressure. Slowly adding ice water and concentrated hydrochloric acid into the residue while stirring, and adjusting the pH to 1-2. Extracting with ethyl acetate, washing the organic phase with saturated salt water, drying with anhydrous sodium sulfate, and concentrating to obtain a crude product of the compound shown in the formula (2) for direct use in subsequent reactions. Wherein, the formula (1) is one selected from methyl p-chloroformyl benzoate, methyl m-chloroformyl benzoate or methyl o-chloroformyl benzoate; the molar ratio of the compound of formula (1) to malononitrile is 1: 1; the molar ratio of the compound of formula (1) to sodium hydride is 1: 2.

(2) And (3) dissolving the product of the formula (2) in tetrahydrofuran, slowly dropwise adding sodium hydroxide dissolved in water at 0 ℃ in an ice bath, removing the ice bath, heating to room temperature, and stirring for reaction for 2-4 h. Tetrahydrofuran in the system was removed by distillation under reduced pressure, water was added, and the mixture was washed with ethyl acetate. And (3) adding hydrochloric acid to adjust the pH value to 1-2, extracting with ethyl acetate, washing an organic phase with saturated salt water, drying with anhydrous sodium sulfate, and concentrating to obtain a crude compound of the formula (3), wherein the crude compound is directly used for subsequent reaction. Wherein the molar ratio of the compound in the formula (2) to the sodium hydroxide is 1 (2-4); the volume ratio of tetrahydrofuran to water in the reaction system is 1 (0.5-2).

(3) The compound of formula (3) is dissolved in acetonitrile and phosphorus pentachloride is added. Stirring and reacting for 2-6 h at 50 ℃ in a nitrogen atmosphere. Cooled to room temperature, and concentrated by distillation under reduced pressure. The residue was dissolved in dichloromethane, washed with water and then saturated brine, dried over anhydrous sodium sulfate and then concentrated to give a pale yellow solid. And dissolving the yellow solid in a mixed solution of acetonitrile and water, and stirring and reacting for 20-60 min at room temperature. Thereafter, ethanethiol and sodium bicarbonate were added in this order. Stirring and reacting for 1-4 h at room temperature, adjusting the pH to 4-5 by using hydrochloric acid, removing acetonitrile in the system by reduced pressure distillation, and then adding saturated salt water. Extracting with ethyl acetate, washing the organic phase with saturated saline solution, drying with anhydrous sodium sulfate, and concentrating to obtain the thiolene ether compound shown in the formula (4). Wherein the molar ratio of the compound in the formula (3) to the phosphorus pentachloride is 1 (2-4); the molar ratio of the compound of formula (3) to ethanethiol and sodium bicarbonate is 1 (1.2-2.4) to 2-6; the volume ratio of the components of the mixed solution of acetonitrile and water is 1:1.

(4) Dissolving the compound of the formula (4) in acetonitrile, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and stirring at room temperature for reaction for 1-2 h. Vacuum distillation and concentration to obtain a crude product of the compound shown in the formula (5), which is directly used for subsequent reaction. Wherein the molar ratio of the compound of formula (4) to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and the N-hydroxysuccinimide is 1 (1-2) to (1-2).

(5) The halogenated amide of linear alkane diamine is dissolved in phosphate buffer (pH 7.0-8.0), the compound of formula (5) dissolved in acetonitrile is added, and the mixture is stirred at room temperature for reaction for 1-2 h. And then adding N-acetyl-L-cysteine and sodium bicarbonate, and stirring and reacting for 1-2 hours at room temperature. Then, the pH was adjusted to 4 to 5 with hydrochloric acid, acetonitrile in the system was removed by distillation under reduced pressure, and then saturated brine was added. Extracting with ethyl acetate, washing the organic phase with saturated saline solution, drying with anhydrous sodium sulfate, and concentrating to obtain the thiolene ether compound shown in the formula (6). Wherein the molar ratio of the compound shown in the formula (5) to the halogenated amide of the straight-chain alkane diamine is 1 (1-2); the volume ratio of the phosphate buffer solution to the acetonitrile in the reaction system is 1 (0.1-1); the molar ratio of the compound of formula (5) to N-acetyl-L-cysteine and sodium bicarbonate is 1 (1-2.5) to 1.2-5.

In a preferred embodiment of the present invention, the reaction time in the step (2) is 2 h; the molar ratio of the compound of formula (2) to sodium hydroxide is 1: 2; the volume ratio of tetrahydrofuran to water in the system is 1:1.

In a preferred embodiment of the present invention, in the step (3), the reaction time of the stirring reaction at 50 ℃ is 4 hours, the reaction time of the stirring reaction at room temperature is 30min, and the reaction time of the stirring reaction at room temperature after the addition of ethanethiol and sodium bicarbonate is 2 hours; the molar ratio of the compound shown in the formula (3) to the phosphorus pentachloride is 1: 3; the molar ratio of the compound of formula (3) to ethanethiol and sodium bicarbonate is 1:1.2: 3.

In a preferred embodiment of the present invention, the reaction time in the step (4) is 1 h; the molar ratio of the compound of formula (4) to 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 1:1.2: 1.2.

In a preferred embodiment of the present invention, the room-temperature stirring reaction time in the step (5) is 1 h; the molar ratio of the compound of formula (5) to the halogenated amide of the linear alkane diamine is 1: 1.2; the volume ratio of the phosphate buffer solution to the acetonitrile in the reaction system is 1:1, and the pH value of the phosphate buffer solution is 7.4; the molar ratio of the compound of formula (5) to N-acetyl-L-cysteine and sodium bicarbonate is 1:2: 3.

In the invention, the thioether compound is applied to polypeptide cyclization, and the thioether compound cyclizes a polypeptide containing a nitrogen-terminal cysteine and an intrachain cysteine so as to construct a cyclic peptide;

specifically, the polypeptide, a thioether compound, N-acetyl-L-cysteine and tris (2-carboxyethyl) phosphine (TCEP) are added into a phosphate buffer solution for reaction, so that the polypeptide is cyclized.

The molar ratio of the polypeptide, the thioether compound, the N-acetyl-L-cysteine and the tri (2-carboxyethyl) phosphine is 1 (1-10) to 1-20; the reaction temperature is 20-40 ℃, and the reaction time is 60-720 min; the pH of the phosphate buffer is 6.0-9.0.

The molar ratio of the polypeptide to the thioether compound to the N-acetyl-L-cysteine to the tris (2-carboxyethyl) phosphine is 1:2:4: 4; the reaction time is 60-120 min; the pH of the phosphate buffer was 7.4.

The nitrogen end of the polypeptide is amino.

The polypeptide is represented by C (Xaa) ac (Xaa) b, C is cysteine (Cys), Xaa is selected from the amino acids glycine (Gly, G), alanine (Ala, a), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), phenylalanine (Phe, P), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), glutamine (Gln, Q), methionine (Met, M), serine (Ser, S), threonine (Thr, T), proline (Pro, P), histidine (His, H), arginine (Arg, R); a is from 1 to 30, b is from 0 to 29, and the sum of a and b is 30.

Performing posttranslational modification on the polypeptide displayed on the surface of the phage in a phosphate buffer solution by using a cyclization method based on the thioether compound, thereby constructing a phage library for displaying cyclic peptide;

specifically, the titer in a phosphate buffer (pH 6.0-9.0) is 10¹³pfu/ml in a phage library of a nitrogen-end displayed polypeptide, adding tris (2-carboxyethyl) phosphine, incubating for 15-60 min at 20-40 ℃, then adding N-acetyl-L-cysteine and the thioether compound, uniformly mixing, incubating for 60-720 min at 20-40 ℃, then adding an ice PEG/NaCl aqueous solution to precipitate phage, placing in ice for 120min, finally obtaining phage precipitate through centrifugation, and resuspending with 1 x phosphate buffer solution to obtain a cyclized phage library; wherein the concentration of TCEP is 0.1-10 mmol/L; thioether compounds and N-acetyl-L-cysteineThe molar ratio of (1-20) to (1-20), the concentration of the thioether compound is 0.1-10 mmol; the PEG/NaCl aqueous solution had the following composition: PEG-8000 (20%, w/w) and sodium chloride (15%, w/w).

In a preferred embodiment of the present invention, in the step of constructing a phage library displaying cyclic peptides, the pH of phosphate buffer is 7.4; the incubation temperature after adding TCEP is 37 ℃, the incubation time is 30min, and the concentration of TCEP is 1 mmol/L; the mol ratio of the thioether compound to the N-acetyl-L-cysteine is 1:4, and the concentration of the thioether compound is 1 mmol/L; the incubation temperature after addition of the thioether compound was 25 ℃ and the incubation time was 120 min.

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

1. the thioether compound can be used for carrying out mild and efficient cyclization on a polypeptide containing a nitrogen-terminal cysteine and an intrachain cysteine to construct a cyclic peptide, and the constructed cyclic peptide can be used for detection, treatment and other applications. In addition, the thioether ether compound can be used for carrying out mild and efficient cyclization on a phage library displaying polypeptide containing one N-terminal cysteine and one in-chain cysteine to construct a cyclized phage library, and the cyclized polypeptide library can be used for screening polypeptides for detection, treatment and other applications aiming at different target proteins. The invention can better synthesize and develop new cyclic peptide with application value.

2. The cyclization method is simple and convenient to operate, has good biocompatibility, can perform cyclization in-vivo and in-vitro environments, is green and economical in used raw materials, does not relate to metal catalysts, is environment-friendly, and accords with the concept of green chemistry.

Drawings

FIG. 1 is a schematic representation of a cyclized molecular cyclized polypeptide and phage library;

FIG. 2 is a flow chart of the phage library circularization efficiency test;

FIG. 3 is a graph comparing cyclization efficiency of phage libraries;

FIG. 4 is a diagram showing the alignment of the phage library to the result of Mdm2 screening;

FIG. 5 is an ELISA test chart of the result of the phage library versus Mdm2 screening.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects of the present invention clearer and clearer, the present invention is further described in detail below with reference to the accompanying drawings and embodiments.