A non-natural amino acid, a preparation method, an aminoacyl tRNA synthetase, a protein or polypeptide containing a non-natural amino acid and applications thereof

Abstract

The present application relates to the field of protein technology, and particularly relates to a non-natural amino acid, a preparation method, an aminoacyl tRNA synthetase, a protein or polypeptide containing the non-natural amino acid and application thereof. The non-natural amino acid is a histidine analogue, and the structural formula of the histidine analogue is one of the following: wherein X is an amino protecting group; Y is a carboxyl protecting group; and A is halogen. The innovation of the present application is that a novel histidine analogue, particularly vinylhistidine (delta VinH), is designed and synthesized, and an aminoacyl tRNA synthetase capable of efficiently recognizing the vinylhistidine is successfully obtained through protein directed evolution technology. The aminoacyl tRNA synthetase can efficiently encode the vinylhistidine into a protein under low concentration (0.1 mM) of the vinylhistidine. The performance of the protein can be significantly improved by introducing the vinylhistidine into the active pocket of a protease or the active center of a biological catalyst through the aminoacyl tRNA synthetase.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of protein technology, and particularly relates to a non-natural amino acid, a preparation method, an aminoacyl tRNA synthetase, a protein or polypeptide containing the non-natural amino acid and application thereof. BACKGROUND

[0002] Histidine is one of the amino acids with the highest frequency of use in enzyme catalytic centers, which can not only act as a nucleophile to participate in the process of hydrolases, but also act as a metal coordination site to regulate the function of metal centers. By regulating the chemical structure of histidine, the chemical diversity of enzyme catalytic centers can be significantly changed, thereby possibly improving the function of enzymes.

[0003] The pKa of natural amino acid side chains is usually in the range of 3-12, which presents different charged states and coordination abilities under physiological conditions, so that different proteins or different regions of the same protein present different physicochemical properties. However, the pKa of almost all amino acid side chains falls outside the physiological pH range (pH = 5-9), which means that the charged state and properties of almost all amino acid side chains are already determined under physiological pH, for example, lysine will be positively charged, and aspartic acid will be negatively charged, which will not change with the fluctuation of physiological pH. The only natural amino acid with a pKa within the physiological pH range is histidine, with a side chain pKa of 7, that is, when the pH of the physiological condition changes from 6 to 8, the histidine side chain will change from 90% positively charged to 10% positively charged. It is this property that enables enzymes to function and proteins to have properties that respond to changes in pH in the range of 6-8.

[0004] However, there is a relatively blank area in the range covered by the pKa of natural amino acids: pH = 4.5-7( Figure 1 ) The lack of this chemical property of natural amino acid side chains means that the function of proteins composed of natural amino acids is still limited, limiting the functional modification of proteins. Taking hydrolases as an example, whether it is an esterase or a protease, the histidine in the catalytic center usually has low activity under physiological acidic conditions (pH = 5-6) such as tumor microenvironment, late endosome, lysosome, etc. This is because the pKa of the histidine side chain is 7, and in the pH = 5-6 range, the histidine in the catalytic center is almost protonated, with poor nucleophilicity, and thus cannot function as a nucleophilic center to exert the function of the enzyme. For example, enzymes with heme as the catalytic center, histidine as the axial ligand of the catalytic center iron atom, is responsible for regulating the electronic properties and catalytic performance of the iron atom. However, regardless of what natural amino acid the axial ligand is mutated to, the change in its chemical structure is a jump, which cannot fine-tune the electronic properties of the catalytic center iron atom, thereby unable to regulate its catalytic performance without changing the catalytic properties of the catalytic center.

[0005] To solve the above problems, the vinyl imidazole functional group of the epsilon position N atom of the vinyl modified imidazole ring is designed, and the pKa of the Nε-hydrogen of histidine is successfully adjusted from 7.07 to 5.71. The pKa of this unnatural amino acid side chain fills the "blank" of the pKa of the natural amino acid side chain in the range of 4.5-7.

[0006] In many catalytic processes involving histidine, the epsilon position N atom (Nε) of the imidazole is usually the active site involved in nucleophilic attack or metal coordination, therefore, Nδ-substituted histidine can be used to regulate the catalytic performance of Nε. By modifying the substituents of the imidazole ring, the performance of the catalytic center can be effectively adjusted, thereby endowing the biological catalyst with new properties. The continuous development of histidine analogs will become a powerful tool for designing and enhancing the activity of artificial enzymes.

[0007] Common enzymes that use histidine as a catalytic center include hydrolytic enzymes and heme-dependent enzymes. In hydrolytic enzymes, histidine is one of the three amino acid residues in the "catalytic triad" and can participate in enzyme function. In heme-dependent enzymes, the iron atom coordinates with the histidine in the enzyme backbone, thereby regulating the catalytic performance of the iron atom.

[0008] Developing new genetically encoded histidine analogs using genetic code expansion technology can greatly improve the catalytic function of enzymes. Genetic code expansion technology is a method that can accurately introduce unnatural amino acids into proteins. By introducing orthogonal aminoacyl tRNA synthetase (aaRS) and tRNA elements, site-specific introduction of unnatural amino acids is achieved. These elements do not interfere with each other and are orthogonal to the endogenous aaRS / tRNA system. The synthetase is responsible for recognizing unnatural amino acids and transferring them to tRNA, while the tRNA recognizes the UAG codon and introduces the unnatural amino acid into the peptide chain. The UAG, which is originally a stop codon, is modified to a codon that encodes an unnatural amino acid, thereby allowing normal gene expression. If the synthetase cannot recognize the unnatural amino acid, gene expression will terminate at UAG. Figure 2 ).

[0009] In this method, genes encoding aaRS and tRNA are introduced into host cells (such as E. coli), the site of interest in the target protein gene is mutated to TAG, and the corresponding unnatural amino acid is added to the culture system, thereby achieving site-specific introduction of unnatural amino acids into the target protein. The key to this process is to evolve aaRS that efficiently recognizes specific unnatural amino acids.

[0010] Esterases are an important class of hydrolytic enzymes, and histidine is usually the catalytic center. However, under physiological acidic conditions (pH = 5-6), natural histidine will be protonated, thereby losing its nucleophilic ability, resulting in weak activity of esterases under acidic conditions.

[0011] Chiral cyclopropane structural units are key components of pharmaceutical molecules, although these structural units can be constructed by organic synthesis methods, but usually require multi-step transformations and low yield. The sperm whale myoglobin carbene transferase (Myoglobin-H64V, V68A) has been reported to be able to catalyze the cyclopropanation reaction of styrene substrates. However, the reaction needs to be carried out in a strict anaerobic environment, and the reaction efficiency of the electron-deficient styrene substrate is low, which limits its universality.

[0012] Myoglobin carbene transferase is a kind of heme-dependent metalloproteinase, and the electronic properties of the heme center iron are regulated by the axial ligand of histidine at position 93. By genetic code expansion technology, replacing the natural histidine with an unnatural histidine axial ligand is expected to regulate the catalytic ability of myoglobin carbene transferase, thereby improving its efficiency and scope of application in cyclopropanation reactions. Nat. Catal. 2018, 1, 578-584. and ACS Catal. 2019, 9, 9683-9697. reported in 2018 and 2019, respectively, that by using pyrrolysine aminoacyl tRNA synthetase mutant (delta MeH-RS) to insert methylhistidine (delta MeH) into His-93 of myoglobin carbene transferase, an artificial metalloenzyme with delta MeH as the catalytic center was successfully obtained. The introduction of delta MeH destroys the original hydrogen bond interaction, reduces the electron-donating ability of histidine, and thereby enhances the activity of myoglobin carbene transferase. However, the construction of the above artificial metalloenzyme needs to rely on high concentration of unnatural amino acid delta MeH (12mM), and the yield of the enzyme is low. These limiting factors hinder the further development of this field.

[0013] Therefore, the prior art still needs to be improved and developed. SUMMARY

[0014] In view of the deficiencies of the prior art described above, the purpose of the present application is to provide a kind of unnatural amino acid, preparation method, aminoacyl tRNA synthetase, protein or polypeptide containing unnatural amino acid and its application, to solve the problem that the natural histidine and methylhistidine (delta MeH) side chain are not acid-resistant, protonation occurs at pH=5-6, and the enzyme function is low under acidic conditions, and to solve the problem that the insertion of methylhistidine in protein needs to rely on high concentration of unnatural amino acid and the insertion efficiency is low.

[0015] The technical scheme of the present application is as follows:

[0016] In the first aspect of the present application, a kind of unnatural amino acid is provided, wherein the unnatural amino acid is histidine analogue, and the structural formula of the histidine analogue is one of the following:

[0017]

[0018] wherein X is an amino protecting group; Y is a carboxyl protecting group; and A is a halogen.

[0019] Optionally, X comprises one of benzyloxycarbonyl, benzyl, 9-fluorenylmethyloxycarbonyl, tert-butyloxycarbonyl and trityl;

[0020] Y comprises one of methyl, ethyl, propyl, isopropyl, benzyl, tert-butyl and trityl;

[0021] A comprises one of Cl, Br and I.

[0022] In a second aspect of the present application, a preparation method of the unnatural amino acid according to the present application is provided, wherein the unnatural amino acid is a histidine analogue, and the preparation method of the histidine analogue comprises:

[0023] Step A, providing histidine, wherein the histidine has a structural formula of wherein ε and δ both represent the nitrogen atom number in the histidine;

[0024] Alternatively, providing histidine whose amino group is protected, histidine whose carboxyl group is protected, or histidine whose amino group and carboxyl group are both protected;

[0025] Step B, protecting the ε-position nitrogen atom of the imidazole ring of the histidine using a protecting group;

[0026] Step C, halogenating the δ-position nitrogen atom of the imidazole ring of the protected histidine through a nucleophilic substitution reaction, and then performing elimination of halogenated hydrocarbon and deprotection to obtain the histidine analogue.

[0027] Optionally, in the step A, the protecting group of the amino group of the histidine is one of benzyloxycarbonyl, benzyl, 9-fluorenylmethyloxycarbonyl, tert-butyloxycarbonyl and trityl;

[0028] The protecting group of the carboxyl group of the histidine is one of methyl, ethyl, propyl, isopropyl, benzyl, tert-butyl and trityl;

[0029] In the step C, the halogen atom is one of Cl, Br and I.

[0030] When the δ-position nitrogen atom of the imidazole ring of the protected histidine is halogenated through a nucleophilic substitution reaction, the raw material used is a triflate of haloethanol.

[0031] Optionally, in the step B, the protecting group of the ε-position nitrogen atom of the histidine is one of benzyloxycarbonyl, benzyl, 9-fluorenylmethyloxycarbonyl, tert-butyloxycarbonyl and trityl,

[0032] Preferably, the protecting group of the epsilon nitrogen atom of the histidine is one of tert-butyloxycarbonyl and trityl,

[0033] More preferably, the protecting group of the epsilon nitrogen atom of the histidine is trityl.

[0034] In a third aspect, the present application provides an aminoacyl tRNA synthetase capable of recognizing the unnatural amino acid according to the present application, wherein the aminoacyl tRNA synthetase is δVinH-RS#1, δVinH-RS#2 or δVinH-RS#3, the sequence of δVinH-RS#1 is shown as SEQ ID NO: 1, the sequence of δVinH-RS#2 is shown as SEQ ID NO: 2, and the sequence of δVinH-RS#3 is shown as SEQ ID NO: 3.

[0035] In a fourth aspect, the present application provides a protein or polypeptide containing an unnatural amino acid, wherein the protein or polypeptide contains an unnatural amino acid, and the structure of the unnatural amino acid in the protein or polypeptide is one or more of the following:

[0036]

[0037] wherein, represents the site of the unnatural amino acid in the protein or polypeptide.

[0038] Optionally, the protein is a hydrolase, and the polypeptide is a polypeptide having a hydrolysis function.

[0039] Optionally, the protein is an esterase, and the polypeptide is a polypeptide having a hydrolysis function.

[0040] Optionally, the protein is a metalloenzyme having histidine as a metal chelating site.

[0041] Optionally, the protein is a heme-dependent metalloprotease.

[0042] In a fifth aspect, the present application provides a use of the protein containing an unnatural amino acid according to the present application as a biological catalyst in biological catalysis.

[0043] Optionally, the protein containing an unnatural amino acid is used as a biological catalyst in catalyzing a hydrolysis reaction.

[0044] Optionally, the protein containing an unnatural amino acid is used as a biological catalyst in catalyzing an ester hydrolysis reaction.

[0045] Optionally, the application of the protein containing unnatural amino acid as a biological catalyst in catalyzing carbene transfer reaction.

[0046] Optionally, the application of the protein containing unnatural amino acid as a biological catalyst in catalyzing nitrogenium transfer reaction.

[0047] Optionally, the application of the protein containing unnatural amino acid as a biological catalyst in catalyzing radical transfer reaction.

[0048] Optionally, the application of the protein containing unnatural amino acid as a biological catalyst in catalyzing cyclopropanation reaction of styrene substrate.

[0049] Beneficial effects: The innovation of the present application lies in designing and synthesizing a brand new histidine analogue, especially vinylhistidine (δVinH), and successfully obtaining an aminoacyl tRNA synthetase capable of efficiently recognizing vinylhistidine through protein directed evolution technology. The aminoacyl tRNA synthetase can introduce δVinH into proteins at a low concentration of δVinH (0.1 mM), which has a significant improvement compared to the insertion efficiency of methylhistidine at this concentration. In addition, introducing δVinH into proteins through the aminoacyl tRNA synthetase can significantly improve the performance of proteins, including esterases and heme-dependent enzymes, etc. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 The design purpose of vinylhistidine (filling the blank of natural amino acid side chain pKa) and application scenario (improving hydrolytic enzymes and metalloenzymes) schematic diagram.

[0051] Figure 2 The basic principle diagram of genetic code expansion technology.

[0052] Figure 3 The analysis diagram of electronic properties of imidazole analogues.

[0053] Figure 4 The chemical structure of vinylhistidine.

[0054] Figure 5 The synthesis route of vinylhistidine.

[0055] Figure 6 The complete synthesis route of vinylhistidine.

[0056] Figure 7 The chemical structure of δVinH characterized by carbon-hydrogen long-range correlation spectroscopy.

[0057] Figure 8 The recognition ability test results of different aminoacyl tRNA synthetases to δVinH.

[0058] Figure 9 The figure shows the test results of δVinHRS#3's ability to recognize different concentrations of δVinH.

[0059] Figure 10 This is the mass spectrometry characterization of GFP-D190δVinH.

[0060] Figure 11 The figure shows the test results of using vinylhistidine to enhance the activity of hydrolases.

[0061] Figure 12 The figure shows the results of Western blotting analysis of the insertion efficiency of methylhistidine and vinylhistidine at the Mb*-H93 site in the myoglobin mutant.

[0062] Figure 13 The mass spectrometry characterization of Mb*-H93δVinH is shown.

[0063] Figure 14 This is a schematic diagram of the cyclopropanation reaction of styrene catalyzed by myoglobin carbene transferase.

[0064] Figure 15 This is a comparison of the results of styrene cyclopropanation under two myoglobin carbene transferases, Mb*-WT-H93 and Mb*-H93δVinH.

[0065] Figure 16 This is a comparison of the results of cyclopropanation of methoxystyrene under two myoglobin carbene transferases, Mb*-WT-H93 and Mb*-H93δVinH.

[0066] Figure 17 This is a comparison of the results of cyclopropanation of 2,3-difluorostyrene under two myoglobin carbene transferases, Mb*-WT-H93 and Mb*-H93δVinH. Detailed Implementation

[0067] This invention provides a non-natural amino acid, its preparation method, an aminoacyl-tRNA synthetase, a protein or polypeptide containing the non-natural amino acid, and its applications. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0068] By analyzing the reason of the catalytic performance improvement of myoglobin carbine transferase, it is found that the enzyme needs a relatively weak electron-donating ability of histidine ligand. By analyzing the imidazole group of the functional side chain of histidine, it is found that it has a more electron-deficient electron-withdrawing imidazole group than natural histidine or methyl histidine (delta MeH), and the pKa of its side chain is 5.71, which provides a new electronic property for histidine Figure 3 ). Therefore, the present application designs and synthesizes a vinyl histidine delta VinH Figure 4 with weak electron-donating ability, which is used to improve the catalytic ability of myoglobin carbine transferase.

[0069] Subsequently, by using protein directed evolution technology, a high-efficiency synthetic enzyme mutant delta VinH-RS which can recognize delta VinH is obtained. The delta VinH-RS can efficiently insert delta VinH at the 190th site of green fluorescent protein (GFP) under low concentration of delta VinH (0.1 mM) to obtain GFP-D190delta VinH, which lays a foundation for the wide application of delta VinH. The delta VinH-RS / delta VinH can be used to efficiently prepare myoglobin carbine transferase (Mb*-H93delta VinH) containing unnatural amino acids, and can efficiently catalyze the cyclopropanation reaction of electron-deficient styrene substrates in aerobic environment. The development of delta VinH-RS / delta VinH greatly improves the catalytic efficiency, reaction conversion rate and oxygen tolerance of myoglobin carbine transferase, and provides a good example for improving biological catalysts by using unnatural amino acids.

[0070] Based on this, the embodiment of the present application provides a kind of unnatural amino acid, wherein the unnatural amino acid is histidine analogue, and the structural formula of the histidine analogue is as shown in one of the following:

[0071]

[0072] Wherein, X is amino protective group;Y is carboxyl protective group;A is halogen.

[0073] In the embodiment of the present application, histidine analogue, especially vinyl histidine delta VinH, is used to improve the performance of protein, especially to improve the catalytic ability of myoglobin carbine transferase and ester hydrolase.

[0074] In one embodiment, X includes but is not limited to one of benzyloxy carbonyl, benzyl, 9-fluorenylmethyloxy carbonyl, tert-butyloxy carbonyl and trityl.

[0075] In one embodiment, Y includes but is not limited to one of methyl, ethyl, propyl, isopropyl, benzyl, tert-butyl and trityl.

[0076] In one embodiment, A comprises one of Cl, Br and I.

[0077] The application provides a preparation method of the unnatural amino acid, wherein the unnatural amino acid is a histidine analogue, and the preparation method of the histidine analogue comprises:

[0078] Step A, providing histidine, and the structural formula of the histidine is wherein ε and δ represent the nitrogen atom numbers in the histidine;

[0079] Alternatively, providing amino-protected histidine, carboxyl-protected histidine or amino- and carboxyl-protected histidine;

[0080] Step B, protecting the ε-position nitrogen atom of the imidazole ring of the histidine by using a protecting group;

[0081] Step C, halogenating the δ-position nitrogen atom of the protected imidazole ring of the histidine by a nucleophilic substitution reaction, and then performing elimination of halogenated hydrocarbon and deprotection reaction to obtain the histidine analogue.

[0082] In one embodiment, the histidine analogue is prepared according to the synthetic route as shown in Figure 5 .

[0083] The side chain of histidine is an imidazole group, two nitrogen atoms in the imidazole group are not equivalent, and there is a rapid tautomerism. If the nitrogen atoms in the imidazole group of the side chain of histidine are derivatized, the main product is the ε-position derivative with less steric hindrance, and the product at this position cannot be coordinated with hematin as a ligand. Therefore, in the embodiments of the application, the ε-position nitrogen atom of the imidazole ring is protected by using a protecting group which is easy to deprotect, and then the δ-position nitrogen atom of the imidazole ring is derivatized, so that the vinyl histidine with the target structure is obtained.

[0084] The protecting group (PG) for protecting the ε-position nitrogen atom of the imidazole ring has multiple choices, for example: benzyloxy carbonyl (Cbz-), benzyl (Bn-), 9-fluorenylmethyloxy carbonyl (Fmoc-), tert-butyloxy carbonyl (Boc-) and trityl (Trt-). Based on the consideration of the deprotection conditions, the Cbz- and Bn- protecting groups need to use metal palladium and dangerous hydrogen when deprotecting, while the Boc- and Trt- protecting groups can be deprotected under mild conditions (weak acid). Therefore, it is preferred to use Boc- and Trt- as the protecting group of the ε-position nitrogen atom of the imidazole ring. In order to use commercially available raw materials, it is further preferred to use the Trt- protecting group to protect the ε-position nitrogen atom of the imidazole ring of the histidine, and then perform halogenation at the δ-position of the imidazole ring by a nucleophilic substitution reaction Figure 6 .

[0085] In one embodiment, the starting material for the halogenation of the delta nitrogen atom of the protected histidine imidazole ring is the triflate of the haloethanol.

[0086] The choice of leaving group (LG) is particularly critical in the nucleophilic substitution reaction. If -Br or -I is chosen as the leaving group, the reaction must be heated to 60°C to react with the nitrogen atom of the imidazole ring. However, experiments have shown that the Trt-protecting group of the epsilon nitrogen of the imidazole ring has been deprotected at 60°C, losing its protective effect. Therefore, the triflate (-OTf) with the strongest leaving ability is chosen, so that the nucleophilic substitution reaction can be carried out quickly at 0°C. The product after the nucleophilic substitution reaction can achieve rapid deprotection of the Trt-protecting group in methanol at 40°C, and then through the elimination of halogenated hydrocarbon and deprotection reaction, the delta-substituted vinyl histidine can be successfully obtained.

[0087] The present application provides an aminoacyl tRNA synthetase capable of recognizing the unnatural amino acid, which is δVinH-RS#1, δVinH-RS#2 or δVinH-RS#3, the sequence of δVinH-RS#1 is shown as SEQ ID NO: 1, the sequence of δVinH-RS#2 is shown as SEQ ID NO: 2, and the sequence of δVinH-RS#3 is shown as SEQ ID NO: 3.

[0088] Since methylhistidine and vinylhistidine have similar structures, the present application constructs a saturation point mutation library based on δMeH-RS, and completes the evolution of the vinylhistidine aminoacyl tRNA synthetase (δVinH-RS) through a negative screening system and a positive screening system. First, the δMeH-RS is subjected to the construction of a multi-site saturation point mutation gene library. Then, the aminoacyl tRNA synthetase recognizing natural amino acids is filtered out through the negative screening system based on the toxic protein Barnase, and then the aminoacyl tRNA synthetase δVinH-RS#1 (L270I, Y271F, L274G, N311D, C313F, Y349F) and δVinH-RS#2 (L270I, Y271F, L274G, N311D, C313Y, Y349F) and δVinH-RS#3 (L270M, Y271L, N311D, C313K, Y349F) recognizing vinylhistidine are successfully screened out through the positive screening system. Among them, δVinH-RS#3 has the highest recognition efficiency for vinylhistidine.

[0089] Specific mutations are: wild-type pyrrolysine aminoacyl tRNA synthetase 270 position is leucine (abbreviated as L), mutated to isoleucine (abbreviated as I) in δVinH-RS#1 and δVinH-RS#2, mutated to methionine (abbreviated as M) in δVinH-RS#3. Wild-type pyrrolysine aminoacyl tRNA synthetase 271 position is tyrosine (abbreviated as Y), mutated to phenylalanine (abbreviated as F) in δVinH-RS#1 and δVinH-RS#2, mutated to leucine (abbreviated as L) in δVinH-RS#3. Wild-type pyrrolysine aminoacyl tRNA synthetase 274 position is leucine (abbreviated as L), mutated to glycine (abbreviated as G) in δVinH-RS#1 and δVinH-RS#2. Wild-type pyrrolysine aminoacyl tRNA synthetase 311 position is asparagine (abbreviated as N), mutated to aspartic acid (abbreviated as D) in δVinH-RS#1, δVinH-RS#2 and δVinH-RS#3. Wild-type pyrrolysine aminoacyl tRNA synthetase 313 position is cysteine (abbreviated as C), mutated to phenylalanine (abbreviated as F) in δVinH-RS#1, mutated to tyrosine (abbreviated as Y) in δVinH-RS#2, mutated to lysine (abbreviated as K) in δVinH-RS#3. Wild-type pyrrolysine aminoacyl tRNA synthetase 349 position is tyrosine (abbreviated as Y), mutated to phenylalanine (abbreviated as F) in δVinH-RS#1, δVinH-RS#2 and δVinH-RS#3.

[0090] The embodiment of the present application provides a protein or polypeptide containing unnatural amino acid, wherein the protein or polypeptide contains unnatural amino acid, and the structural formula of the unnatural amino acid in the protein or polypeptide is one or more of the following:

[0091]

[0092] wherein, indicates the connection site of the unnatural amino acid in the protein or polypeptide.

[0093] Further, the protein is a hydrolase, and the polypeptide is a polypeptide with hydrolysis function.

[0094] Further, the protein is an esterase, and the polypeptide is a polypeptide with ester hydrolysis function.

[0095] Further, the protein is a metal enzyme with histidine as a metal chelation site.

[0096] Further, the protein is a heme-dependent metalloprotease.

[0097] The introduction of delta VinH into the heme-dependent metalloprotease by aminoacyl tRNA synthetase can significantly improve the catalytic performance and the reaction range of the heme-dependent metalloprotease.

[0098] Further, the heme-dependent metalloprotease is myoglobin carbene transferase, and the TAG amino acid at position 93 of the myoglobin carbene transferase is mutated to the vinyl histidine.

[0099] The introduction of delta VinH into the 93 position of the myoglobin carbene transferase by aminoacyl tRNA synthetase can significantly improve the oxygen tolerance and the reaction activity of the myoglobin carbene transferase to the electron-deficient styrene.

[0100] The application provides an application of the protein containing the unnatural amino acid as a biological catalyst in biological catalysis.

[0101] Further, the application of the protein containing the unnatural amino acid as a biological catalyst in catalyzing the hydrolysis reaction.

[0102] Further, the application of the protein containing the unnatural amino acid as a biological catalyst in catalyzing the ester hydrolysis reaction.

[0103] Further, the application of the protein containing the unnatural amino acid as a biological catalyst in catalyzing the carbene transfer reaction.

[0104] Further, the application of the protein containing the unnatural amino acid as a biological catalyst in catalyzing the nitrogenium transfer reaction.

[0105] Further, the application of the protein containing the unnatural amino acid as a biological catalyst in catalyzing the radical transfer reaction.

[0106] Further, the application of the protein containing the unnatural amino acid as a biological catalyst in catalyzing the cyclopropanation reaction of the styrene substrate.

[0107] The application is further described below through specific examples.

[0108] Example 1: Synthesis of vinyl histidine

[0109] Combination Figure 6 As shown in the figure, the synthesis steps of the vinyl histidine are as follows:

[0110] Commercially available compound Boc-His(Trt)-OH (20 mmol, 1 eq), N,N dimethyl amino pyridine DMAP (20 mmol, 1 eq), dicyclohexyl carbodiimide DCC (20 mmol, 1.1 eq) were taken in a 500 mL round bottom flask. Then 100 mL dichloromethane (DCM) and 100 mL methanol (MeOH) were added and the reaction was stirred at room temperature for 12 hours, during which white precipitate was formed. After confirming the completion of the reaction by thin layer chromatography (TLC) test, the reaction was terminated. After spinning off the solvents using a rotary evaporator, 100 mL ethyl acetate was added and filtered under reduced pressure to remove the white precipitate. The filtrate was concentrated using a rotary evaporator and the final product was isolated by silica gel column chromatography (ethyl acetate / pet ether) to obtain the target compound 1 as a white solid after drying with ethyl ether, with a yield of 90%.

[0111] Nuclear magnetic resonance hydrogen spectrum data: 1 H NMR (400 MHz, CDC13) δ 7.43 - 7.28 (m, 10H), 7.15 - 7.05 (m, 6H), 6.53 (s, 1H), 5.99 (d, J = 8.4 Hz, 1H), 4.52 (m, 1H), 3.59 (s, 3H), 3.04 (dd, J = 14.6, 5.2 Hz, 1H), 2.97 (dd, J = 14.4, 4.8 Hz, 1H), 1.41 (s, 9H). 3 C NMR (101 MHz, CDC13) δ 155.67, 142.33, 138.76, 136.48, 129.84, 128.17, 128.15, 119.69, 79.61, 75.40, 53.83, 52.11, 30.34, 28.44. + Predicted: 512.2549, Found: 512.2533.

[0112] The compound 1 obtained in the previous step (10 mmol, 1 eq) was placed in a dry 500 mL round bottom flask under nitrogen protection, followed by the addition of 100 mL ultra-dry dichloromethane (DCM). The reaction system was cooled in an ice water bath for 10 minutes, and then 2-bromoethyl triflate (11 mmol, 1.1 eq) was slowly added dropwise. After the addition was completed, the reaction system was reacted at 0°C overnight (about 12 hours). After confirming the completion of the reaction by thin layer chromatography (TLC) test, the dichloromethane was spun off. 100 mL of methanol was added to completely dissolve the product, and the reaction was continued at 40°C for 4 hours. After the deprotection reaction was completed, the methanol was spun off using a rotary evaporator, and separation and purification were performed by silica gel column chromatography (ethyl acetate / pet ether) to obtain the target compound 2 as a yellow oily liquid, with a yield of 54%.

[0113] NMR hydrogen spectrum data: 1 H NMR (400 MHz, CDC13) δ 7.99 (s, 1H), 6.89 (s, 1H), 5.29 (d, J = 7.6 Hz, 1H), 4.53 (m, 1H), 4.47-4.28 (m, 1H), 3.76 (s, 2H), 3.62 (t, J = 6.5 Hz, 2H), 3.18 (dd, J = 15.7, 5.7 Hz, 1H), 3.11 (dd, J = 15.6, 5.9 Hz, 1H), 1.42 (s, 9H). NMR carbon spectrum data: 13 CNMR (101 MHz, CDC13) δ 171.42, 155.35, 137.82, 127.70, 125.67, 80.64, 53.09, 52.92, 46.64, 30.05, 28.35, 26.78. High resolution mass spectrum [M+H] + Predicted: 376.0872, Found: 376.0862.

[0114] The compound 2 obtained in the previous step (5 mmol, 1 eq) was placed in a dry 500 mL round bottom flask under nitrogen protection, anhydrous potassium carbonate solid (25 mmol, 5 eq) was added, and finally 50 mL of ultra-dry dimethylformamide (DMF) was added. Reaction overnight (about 12 hours) at 60°C, the reaction was detected by thin layer chromatography (TLC). Add 200 mL of deionized water to terminate the reaction, extract with ethyl acetate, wash with saturated brine, and dry with anhydrous sodium sulfate. The solvent was spin-dried with a rotary evaporator, and the target compound 3 was obtained by silica gel column chromatography (ethyl acetate / petroleum ether) for separation and purification, with a yield of 47% as a light yellow oily liquid.

[0115] NMR hydrogen spectrum data: 1 H NMR (400 MHz, CDC13) δ 7.99 (s, 1H), 6.89 (s, 1H), 5.29 (d, J = 7.6 Hz, 1H), 4.53 (m, 1H), 4.47-4.28 (m, 1H), 3.76 (s, 2H), 3.62 (t, J = 6.5 Hz, 2H), 3.18 (dd, J = 15.7, 5.7 Hz, 1H), 3.11 (dd, J = 15.6, 5.9 Hz, 1H), 1.42 (s, 9H). NMR carbon spectrum data: 13C10 NMR (101 MHz, CDCl3) δ 171.28, 154.89, 134.53, 128.40, 127.78, 125.70, 104.63, 79.53, 52.82, 52.10, 27.94, 26.29. High-resolution mass spectrometry [M+H] + Predicted value: 296.1610, Experimental value: 296.1601.

[0116] Compound 3 (2 mmol, 1 eq) obtained in the previous step was dissolved in 10 mL of 1,4-dioxane solvent, and 10 mL of 5N sodium hydroxide solution was added. The reaction was carried out at room temperature for 1 hour. After the reaction was confirmed to be complete by thin-layer chromatography (TLC), the pH was adjusted to neutral (pH = 7) with 6N hydrochloric acid, and the solvent was evaporated. Compound 4 was then separated by C18 reversed-phase column chromatography. Compound 4 was then dissolved completely in 20 mL of dichloromethane (DCM), and then slowly added dropwise to 20 mL of 4N hydrogen chloride-diethyl ether solution. After reacting for 30 minutes, a large amount of white precipitate was formed. The precipitate was filtered to obtain vinylhistidine dihydrochloride as a white solid, with a yield of 69%.

[0117] 1H NMR spectroscopy data: 1 ¹H NMR (400MHz, MeOD) δ 9.35 (s, 1H), 7.68 (s, 1H), 7.28 (dd, J = 15.2, 8.4 Hz, 1H), 6.00 (dd, J = 15.2, 2.1 Hz, 1H), 5.69 (dd, J = 8.4, 2.2 Hz, 1H), 4.38 (t, J = 7.1 Hz, 1H), 3.62–3.45 (m, 2H). Carbon NMR data: 13 C10 NMR (101 MHz, MeOD) δ 168.61, 134.40, 128.12, 126.85, 119.58, 115.05, 50.95, 23.96, High-resolution mass spectrometry [M+H] + Predicted value: 182.0925, Experimental value: 182.0923.

[0118] Example 2: NMR characterization of vinylhistidine

[0119] To prove that it passed Figure 6 The synthesized vinylhistidine obtained is δ-substituted rather than ε-substituted. The obtained vinylhistidine was characterized in detail using nuclear magnetic resonance (NMR) spectroscopy. Specifically, the assignment of each signal peak was determined using 1H NMR, 1C NMR, H-H correlation spectroscopy, and C-H correlation spectroscopy. Finally, H-H correlation was observed using HMBC spectroscopy. a and C b And C aThere are relevant signals ( Figure 7 Therefore, it was determined that the obtained product was δ-substituted vinylhistidine δVinH.

[0120] Example 3: Evolution of vinylhistidine aminoacyl-tRNA synthetase

[0121] Because methylhistidine and vinylhistidine have similar structures, this embodiment constructed a multi-site saturated point mutation library based on δMeH-RS, and completed the evolution of vinylhistidine aminoacyl-tRNA synthetase (δVinH-RS) using negative and positive selection systems. A negative selection system based on the toxic protein Barnase was used to filter out synthase mutants that recognize native amino acids. Then, a positive selection system successfully identified the aminoacyl-tRNA synthetases δVinH-RS#1 (270I, 271F, 274G, 311D, 349F), δVinH-RS#2 (270I, 271F, 274G, 311D, 313Y, 349F), and δVinH-RS#3 (270M, 271L, 311D, 313K, 349F) that recognize vinylhistidine.

[0122] Example 4: Efficiency characterization of vinylhistidine aminoacyl-tRNA synthetase in Example 3

[0123] This embodiment uses GFP-D190TAG as a model protein to compare the recognition efficiency of δVinH-RS#(1-3) for vinylhistidine. The fluorescence intensity of GFP shows that δVinH-RS#3 has the highest recognition ability for vinylhistidine, and the expression level of GFP-D190δVinH reaches four-fifths of the expression level of wild-type green fluorescent protein GFP-WT. Figure 8 As shown, Figure 8 In the diagram, Hit 1# represents δVinH-RS#1, Hit 2# represents δVinH-RS#2, and Hit 3# represents δVinH-RS#3. Subsequently, tests were conducted using δVinH-RS#3 at different vinylhistidine concentrations. It was found that under the condition of 0.1 mM vinylhistidine, the expression level of δVinH in GFP-D190 was half that of GFP-WT, demonstrating that δVinH-RS#3 has extremely high recognition efficiency for δVinH. Figure 9 Mass spectrometry analysis further confirmed that δVinH was successfully introduced into the 190 site of GFP. Figure 10 Then, the recognition of vinyl histidine by δVinH-RS (δVinH, 1mM) and the recognition of methyl histidine by δMeH-RS (δMeH, 1mM) were compared. The fluorescence intensity of green fluorescent protein demonstrated that the ability of δVinH-RS to recognize vinyl histidine was four times that of δMeH-RS to recognize methyl histidine.Figure 11 ).

[0124] Example 5: Preparation and characterization of ester hydrolysis enzyme OE1.3-H23δVinH

[0125] OE1.3-H23δVinH protein was purified by transforming δVinH-RS#3 gene and OE1.3-H23TAG-StrepTagII gene in Example 3 into E. coli DH10b competent cells, inducing expression in LB medium containing 1 mM δVinH, and purifying OE1.3-H23δVinH protein in a similar manner to purify OE1.3-H23δMeH and OE1.3-H23H proteins. By performing ester hydrolysis reaction on the substrate under the condition of pH = 5.5, the product after hydrolysis will produce fluorescence, and the degree of catalytic reaction can be detected by fluorescence change. Experimental results prove that the catalytic ability of OE1.3-H23δVinH is higher than that of OE1.3-H23δMeH and OE1.3-H23H under the condition of pH = 5.5 Figure 11 ).

[0126] Example 6: Preparation and characterization of myoglobin carbonyl transferase Mb*-H93δVinH

[0127] By transforming δVinH-RS#3 gene and Mb*-H93TAG-HisTag gene in Example 3 into E. coli DH10b competent cells, expression was induced in LB medium containing 1 mM δVinH. Western-Blot analysis showed that δVinH was successfully introduced into the 93 position of Mb* (Myoglobin). By comparing with the expression of Mb*-WT-HisTag and Mb*-H93δMeH-HisTag, it was proved that the expression amount of Mb*-H93δVinH was close to the wild type, and was much higher than that of Mb*-H93δMeH Figure 12 ). Mass spectrometry analysis further confirmed that δVinH was successfully introduced into the 93 site of Mb* Figure 13 ).

[0128] Two kinds of myoglobin carbonyl transferases, Mb*-WT and Mb*-H93δVinH, were purified for testing the cyclopropanation reaction of styrene Figure 14-15 ), and selected styrene Figure 14 ), p-methoxystyrene Figure 16 ) and electron-deficient 2,3-difluorostyrene Figure 17) as substrates. The catalytic reaction was carried out in air, and the quantitative analysis was performed by HPLC after 5 minutes. The results showed that Mb*-WT exhibited low activity for the above three substrates, with a conversion rate of 30% for substrate 1a, 53% for substrate 2a, and 16% for substrate 3a. However, Mb*-H94δVinH exhibited extremely high activity for these substrates, with a conversion rate of 78% for substrate 1a, 79% for substrate 2a, and 80% for substrate 3a. This indicates that the introduction of δVinH into the 93 site of Mb* not only improves the oxygen tolerance of myoglobin carbene transferase, but also enhances its reactivity for electron-deficient styrene.

[0129] It should be understood that the application of the present application is not limited to the above examples, and can be improved or changed according to the above description for those of ordinary skill in the art, and all such improvements and changes shall fall within the scope of protection of the appended claims of the present application.

Claims

1. A non-natural amino acid, characterized in that, The unnatural amino acid is a histidine analogue, and the structural formula of the histidine analogue is as follows:

2. A method of producing the unnatural amino acid of claim 1, wherein The unnatural amino acid is a histidine analogue, and the preparation method of the histidine analogue comprises: Step A, providing histidine having the structural formula wherein both ε and δ represent the nitrogen atom numbering in histidine; Alternatively, an amino-protected histidine, a carboxyl-protected histidine, or a histidine with both amino and carboxyl groups protected is provided; Step B: protecting the epsilon nitrogen atom of the imidazole ring of the histidine using a protecting group; Step C: haloethylation of the delta nitrogen atom of the protected histidine imidazole ring through a nucleophilic substitution reaction, followed by elimination of the haloalkane and deprotection to obtain the histidine analogue; In step A, the protecting group of the amino group of the histidine is one of benzyloxy carbonyl, benzyl, 9-fluorenylmethyloxy carbonyl, tert-butyloxy carbonyl, and trityl; The protecting group of the carboxyl group of the histidine is one of methyl, ethyl, propyl, isopropyl, benzyl, tert-butyl, and trityl; In step C, the halogen atom is one of Cl, Br, and I; When haloethylation of the delta nitrogen atom of the protected histidine imidazole ring is performed through a nucleophilic substitution reaction, the raw material used is a triflate of haloethanol; In step B, the protecting group of the epsilon nitrogen atom of the histidine is one of benzyloxy carbonyl, benzyl, 9-fluorenylmethyloxy carbonyl, tert-butyloxy carbonyl, and trityl.

3. The method for preparing non-natural amino acids according to claim 2, characterized in that, The protecting group of the epsilon nitrogen atom of the histidine is one of tert-butyloxy carbonyl and trityl.

4. The method of producing a non-natural amino acid according to claim 3, wherein The protecting group of the epsilon nitrogen atom of the histidine is trityl.

5. An aminoacyl tRNA synthetase capable of recognizing the unnatural amino acid of claim 1, wherein the aminoacyl tRNA synthetase is δVinH-RS#1, δVinH-RS#2, or δVinH-RS#3, the sequence of δVinH-RS#1 is shown as SEQ ID NO: 1, the sequence of δVinH-RS#2 is shown as SEQ ID NO: 2, and the sequence of δVinH-RS#3 is shown as SEQ ID NO:

3.

6. A protein or polypeptide containing non-natural amino acids, characterized in that, The protein or polypeptide contains an unnatural amino acid, and the structural formula of the unnatural amino acid in the protein or polypeptide is as follows: wherein, represents the site of attachment of the unnatural amino acid in the protein or polypeptide.

7. The protein or polypeptide containing unnatural amino acid according to claim 6, wherein The protein is a hydrolase or an esterase; Alternatively, the polypeptide is a polypeptide with hydrolysis function or a polypeptide with hydrolysis ester function; Alternatively, the protein is a metalloenzyme with histidine as a metal chelation site; Alternatively, the protein is a heme-dependent metalloprotease.

8. Use of the protein containing the unnatural amino acid of any one of claims 6-7 as a biological catalyst in biological catalysis.

9. Use according to claim 8, characterized in that, The protein containing the unnatural amino acid is used as a biological catalyst in catalyzing a carbene transfer reaction, a nitrene transfer reaction, a radical transfer reaction, or a hydrolysis reaction.

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