A non-natural amino acid based on thiocoumarin and its preparation method and application

Abstract

The present invention relates to a non-natural amino acid based on thiocoumarin and a preparation method and application thereof. The compound non-natural amino acid (SCouK) developed by the present invention can be used for site-specific introduction into target proteins in prokaryotic and eukaryotic systems to regulate their activity. The important significance of the present invention is that it provides a method with advantages such as longer and wider wavelength of light uncaging, higher uncaging efficiency, lower cytotoxicity, and better biocompatibility. It has good potential application prospects in the light activation regulation of proteins with lysine as a key residue, and can provide an alternative and preferred tool for protein function regulation.

Description

Technical Field

[0001] The present invention relates to the field of protein activity regulation, and in particular to a non-natural amino acid based on thiocoumarin and a preparation method and application thereof. Technical Background

[0002] As the main material basis of life activities and the executor of biological functions, proteins play an indispensable and important role in almost all biological processes, such as metabolism, signal transduction, homeostasis and cell division. Precise regulation of protein function and activity at the molecular level and direct observation of the resulting changes in biological systems are crucial to gaining a deeper understanding of protein functions and the roles they play in life activities.

[0003] In recent years, the emerging bioorthogonal cleavage reaction (BCR) has gradually become a powerful tool for manipulating biomolecules and studying gain of function in living systems. BCR based on unnatural amino acids has been widely used in the functional regulation and in situ activation of proteins in biological systems. Through genetic codon expansion technology, the key amino acid residues of proteins are replaced with unnatural amino acids protected by chemical cages or photocage groups, so that the activity of proteins can be temporarily "turned off"; BCR mediated by light, metals or small molecules is used to remove the protecting groups and restore them to key amino acids, and then "turned on" in situ, thereby achieving precise regulation of protein function. Compared with traditional functional regulation methods, this protein activation strategy based on unnatural amino acids shows superior temporal and spatial precision and site precision, especially providing a unique means for studying the function of specific sites of target proteins.

[0004] Although great progress has been made in chemical caged unnatural amino acids based on BCR, photocaged unnatural amino acids are still the most widely used and most popular tool for regulating protein site-specific activity. This is mainly because light has significant advantages such as non-invasiveness, uniform distribution and easy control, and can control the release of protein active sites in living cells and in vivo with high spatiotemporal resolution, thereby regulating the function of proteins. Photocaged unnatural amino acids are usually used to site-specifically replace key functional residues of proteins, temporarily "turning off" protein activity; when light triggers the removal of photocaged protective groups, the activity of proteins is "turned on", thereby achieving precise control of protein function from "off" to "on". However, most of the existing photocaged unnatural amino acids are based on o-nitrobenzyl photocage groups, which require high-energy ultraviolet light for photodecagement reactions, and ultraviolet light has biological toxicity and low penetration. In addition, the decagement efficiency of existing photocaged unnatural amino acids is low, and the problem of toxic byproducts after decagement also needs to be solved. Therefore, it is urgent to develop photocaged unnatural amino acids with longer decagement wavelengths and higher decagement efficiencies. Summary of the invention

[0005] In order to improve the shortcomings of existing photocaged unnatural amino acids, thiocoumarin-based photocaged groups can be removed by blue light or green light, and have the characteristics of small size, simple synthesis, high uncaging efficiency, low cytotoxicity, etc., and have been used for the controlled release of oligonucleotides and small molecule ligands. Therefore, the purpose of the present invention is to provide a thiocoumarin-based unnatural amino acid (a lysine analogue), which undergoes efficient uncaging reaction mediated by green light, and can be inserted into the target protein by genetic codon expansion technology, and used for light-controlled activation of the target protein in living cells, thereby Figure 1 The design purpose of the present invention can be seen in FIG.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a type of light-caged non-natural amino acid SCouK that can regulate protein activity reactions, and its structural formula is as follows:

[0008]

[0009] Specifically, its structure contains lysine residues as well as thiocoumarins.

[0010] The present invention also provides a chemical synthesis method of a photocaged unnatural amino acid SCouK, comprising the following steps:

[0011] Step 1) using resorcinol as a starting material, condensing and cyclizing with ethyl 4-chloroacetoacetate under acidic conditions to obtain compound A (3-1);

[0012] Step 2) hydrolyzing compound A to produce compound B (3-2);

[0013] Step 3) protecting the dihydroxyl group of compound B (3-2) with acetyl to obtain compound C (3-3);

[0014] Step 4) The obtained compound C (3-3) is subjected to a thiolation reaction in the dark to generate compound D (3-4);

[0015] Step 5) removing the acetyl group from the obtained compound D (3-4) with hydrochloric acid to obtain compound E (3-5);

[0016] Step 6) reacting compound E (3-5) with p-nitrophenyl chloroformate to obtain an activated ester compound F (3-6) of thiocoumarin;

[0017] Step 7) The activated ester compound F (3-6) obtained is connected with Nα-tert-butyloxycarbonyl-L-lysine to generate compound G (3-7)

[0018] Step 8) Remove the tert-butyloxycarbonyl (Boc) group with trifluoroacetic acid to obtain the photocaged unnatural amino acid (SCouK)

[0019] The present invention also provides a compound and a composition connected with the non-natural amino acid.

[0020] The present invention also provides a protein expression system, a translation system and a cell comprising the non-natural amino acid.

[0021] The present invention also provides the use of the non-natural amino acid in site-specific introduction into proteins in prokaryotic and eukaryotic systems to regulate their activity.

[0022] The present invention also provides application of the non-natural amino acid in enhancing the activation of the fluorescence signal of green fluorescent protein (EGFP).

[0023] The present invention also provides the application of the non-natural amino acid in protein kinase activity.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1) Based on the thiocoumarin photocage group, a photocage non-natural amino acid SCouK was designed and synthesized, and it was site-specifically inserted into the key lysine site of the target protein using genetic codon expansion technology, rendering it inactive. Subsequently, the thiocoumarin group was removed through a green light-mediated photodecagement reaction to regenerate natural lysine and restore the activity of the protein, thereby achieving functional and activity regulation of multiple target proteins in living cells.

[0026] 2) This study used HPLC-MS to detect the reaction products of Fmoc-SCouK after green light irradiation at the amino acid level, verifying that Fmoc-SCouK can be rapidly dethiocoumarinized by green light to release FmocK. In order to introduce SCouK into the target protein, this study screened the synthetase mutant SCouKRS that efficiently recognizes SCouK, and introduced SCouK into the model protein sfGFP in Escherichia coli and mammalian cells through genetic codon expansion technology. Using the recombinantly expressed and purified sfGFP-N150SCouK, this study found that the protein could generate sfGFP-N150K after green light irradiation, proving the feasibility of SCouK photocage at the protein level.

[0027] 3) SCouK can be used to activate the fluorescence signal of EGFP. Through the exploration of different wavelengths and different durations of illumination conditions, this study found that SCouK can effectively activate the fluorescence signal of EGFP in a very short time (about 1 minute) by 530nm green light irradiation.

[0028] 4) SCouK was applied to regulate the activity of proteins with different catalytic functions, including fLuc, MEK1, and Src. By introducing SCouK into the lysine sites related to the catalytic activity of these proteins, this study achieved rapid regulation of fLuc bioluminescence catalytic activity, MEK1 kinase activity, and Src autophosphorylation and substrate protein phosphorylation activity in living cells.

[0029] 5) The newly developed photocaged unnatural amino acid SCouK in the present invention has the advantages of longer and wider wavelength of photodecagement, higher decagement efficiency, lower cytotoxicity, and better biocompatibility. It has good potential application prospects in the photoactivated regulation of proteins with lysine as the key residue, and can provide an alternative and preferred tool for protein function regulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The drawings described herein are used to provide a further understanding of the present invention, constitute a part of the present application, and do not constitute an improper limitation of the present invention. In the drawings:

[0031] Figure 1 :Schematic diagram of the strategy of using thiocoumarin-based photocaged unnatural amino acids to regulate protein activity;

[0032] Figure 2 : Schematic diagram of SCouK light detachment;

[0033] Figure 3 : SCouK synthesis route map;

[0034] Figure 4 :Analysis diagram of photo-uncaging reaction of Fmoc-SCouK, (A) Structure of Fmoc-SCouK and photo-uncaging reaction; (B)

[0035] HPLC-MS analysis of Fmoc-SCouK photodecaging reaction; detector wavelength: 254 nm; (C) The molecular weight of SCou-OH corresponding to the chromatographic peak at retention time 9.2 min; (D) Relative quantification and conversion analysis based on the chromatographic peak areas of Fmoc-SCouK and FmocK in (B);

[0036] Figure 5 : Results of site-directed insertion of SCouK into target proteins in E. coli; (A) Screening results of MmPylRS synthetase mutants that recognize SCouK; (B) Site-directed introduction of SCouK into sfGFP-N150TAG protein in E. coli; (C) Expression of sfGFP-N150SCouK protein under different concentrations of SCouK in E. coli. CBB: Coomassie Brilliant Blue;

[0037] Figure 6:Analysis of the molecular mass spectra of sfGFP-N150SCouK before and after illumination; (A) sfGFP-N150SCouK before illumination; (B) sfGFP-N150SCouK after illumination. Left: Mass spectrum after deconvolution; Right: Mass spectrum before deconvolution;

[0038] Figure 7 : Results of site-specific introduction of sfGFP-N150TAG protein by large SCouK in mammalian cells. (A) Western blot analysis of site-specific introduction of sfGFP-N150TAG by SCouK in HEK293T cells; (B) Western blot analysis of site-specific introduction of sfGFP-N150TAG by SCouK at different concentrations in HEK293T cells; (C) Fluorescence imaging analysis of site-specific introduction of sfGFP-N150TAG by SCouK at different concentrations in HEK293T cells; Scale bar: 100 μm;

[0039] Figure 8 :Schematic diagram of SCouK light uncaging reaction activating EGFP-K85SCouK-mCherry fluorescence signal;

[0040] Fig. 9 :Confocal fluorescence imaging of EGFP-K85SCouK-mCherry in living cells after illumination with different wavelengths. (A) Confocal fluorescence imaging analysis of the effect of illumination with different wavelengths on the fluorescence signal of EGFP-K85SCouK-mCherry; Scale bar: 100 μm; (B) Quantitative analysis of the relative fluorescence intensity ratio of EGFP / mCherry in (A); Data are shown as mean ± standard deviation (n = 5); Blue is DAPI staining 1;

[0041] Fig.10 :Confocal fluorescence imaging of EGFP-K85SCouK-mCherry in living cells after different illumination times. (A) Confocal fluorescence imaging analysis of the effect of different illumination durations on the fluorescence signal of EGFP-K85SCouK-mCherry; Scale bar: 100μm. (B) Quantitative analysis of the relative fluorescence intensity ratio of EGFP / mCherry in (A); Data are shown as mean ± SD (n = 4);

[0042] Fig.11 :Schematic diagram of SCouK used to regulate luciferase activity;

[0043] Fig.12: Schematic diagram of light activation in HEK293T cells. (A) HCK structure; (B) Bioluminescence quantitative detection of the activation of fLuc-K529UAA luciferase activity by light of different wavelengths; data are shown as mean ± standard deviation (n = 3); (C) Analysis of the expression of fLuc-K529HCK and fLuc-K529SCouK; (D) Bioluminescence imaging and immunoblot analysis of fLuc-K529SCouK activated by light of different wavelengths;

[0044] Fig.13 :SCouK is used for photoactivation of MEK1. (A) Schematic diagram of the partial Raf / MEK / ERK signaling pathway activated by light

[151] ; (B) Photoactivated MEK1-ΔN-K97SCouK in HEK293T cells regulates downstream ERK phosphorylation levels; anti-p-ERK1 / 2: ERK1 / 2 phosphorylation antibody;

[0045] Fig.14 : Schematic diagram of SCouK for Src photoactivation. (A) Schematic diagram of Src photoactivation, "C" represents the photocaged unnatural amino acid, and "P" represents phosphorylation modification; (B) Light-activated Src-Y527F-K295SCouK in HEK293T cells regulates Src autophosphorylation and downstream substrate protein tyrosine phosphorylation levels. DETAILED DESCRIPTION

[0046] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased from the market.

[0047] Example 1: Design and synthesis of the unnatural amino acid SCouK

[0048] according to Figure 1 In this study, a photocaged unnatural amino acid SCouK was designed by connecting thiocoumarin to lysine via carbamate. Figure 1 The general structural formula described in the above, its cage-release principle is as follows Figure 2 As shown, after the thiocoumarin photocage group absorbs photons of a certain wavelength, the chemical bond breaks, generating an ion pair of thiocoumarin and lysine carbonamide, in which the positive charge is located in the former and the negative charge is located in the latter. The former is further hydrolyzed to form 4-hydroxymethyl-7-hydroxythiocoumarin (SCou-OH), while the latter decomposes to obtain lysine by eliminating a molecule of carbon dioxide.

[0049] The synthetic route of the unnatural amino acid SCouK is as follows Figure 3 shown.

[0050]

[0051] Compound A Synthesis

[0052] Resorcinol (10 g, 91.0 mmol) was dissolved in 70% sulfuric acid solution (100 mL). After the solution was cooled to 0°C, ethyl 4-chloroacetoacetate (12.5 mL, 91.0 mmol) was added dropwise thereto. After the reaction solution was warmed to room temperature, it was stirred overnight. After the reaction was completed, the reaction solution was poured into 500 mL of ice water, the precipitate was filtered, washed with ice water, and dried to obtain a white solid 3-1 (10.7 g, 56%). 1 H NMR (400MHz, CDCl3) δ7.52(d,J=8.6Hz,1H),6.84(dd,J=8.6,2.4Hz,1H),6.82(d,J=2.4Hz,1H),6.37(s,1H),4.63(s,2H). 1 H NMR analysis was consistent with that reported in the literature.

[0053] Compound B Synthesis

[0054] 3-1 (10.7 g, 51.0 mmol) was added to deionized water (400 mL) and heated under reflux for 48 hours. After the reaction was completed, the reaction solution was cooled to room temperature and then placed in a refrigerator at 4°C for 24 hours to precipitate. The precipitate was filtered, washed with ice water, and dried to obtain a white solid 3-2 (6.6 g, 67%). 1 H NMR(500MHz,,CD3OD)δ7.50(d,J=8.7Hz,1H),6.79(dd,J=8.7,2.4Hz,1H),6.72(d,J=2.4Hz,1H),6.36(s,1H),4.81(d,J=1.6Hz,2H). 1 HNMR analysis was consistent with that reported in the literature.

[0055] Compound C Synthesis

[0056] 3-2 (6.6 g, 34.1 mmol) was dissolved in anhydrous THF (100 mL), and acetic anhydride (19.0 mL, 204.8 mmol), pyridine (2.76 mL, 34.1 mmol) and DMAP (417 mg, 3.41 mmol) were added to the solution in sequence, and stirred at room temperature for 2 hr. After the reaction was completed, the reaction solution was concentrated under reduced pressure, ethyl acetate was added and washed with water, dried and concentrated, and purified by column chromatography (petroleum ether / ethyl acetate = 5 / 1) to obtain a white solid 3-3 (7.7 g, 82%). 1 H NMR (400MHz, CDCl3) δ7.53(d,J=8.7Hz,1H),7.17(d,J=2.3Hz,1H),7.10(dd,J= 8.6, 2.3Hz, 1H), 6.48 (s, 1H), 5.28 (d, J = 1.5Hz, 2H), 2.35 (s, 3H), 2.22 (s, 3H). 13 HRMScalcd for C 14 H 12 O6[M+H] + :277.0707; found:277.0708.

[0057] Compound D Synthesis

[0058] 3-3 (7.7 g, 28.0 mmol) and Lawesson's reagent (9.1 g, 22.4 mmol) were added to anhydrous m-xylene (150 mL) and heated under reflux for 6 hr in the dark. After the reaction was completed, the reaction solution was concentrated under reduced pressure and purified by column chromatography (petroleum ether / ethyl acetate = 5 / 1) to obtain an orange solid 3-4 (5.6 g, 69%). 1 H NMR (400MHz, CDCl3) δ7.55(d,J=8.7Hz,1H),7.29(d,J=2.2Hz,1H),7.26(s,1H) ,7.13(dd,J=8.7,2.2Hz,1H),5.21(d,J=1.3Hz,2H),2.35(s,3H),2.20(s,3H). 13HRMS calcd for C 14 H 112 O5S[M+H] + :293.0478; found:293.0478.

[0059] Compound E Synthesis

[0060] 3-4 (5.6 g, 19.3 mmol) was dissolved in ethanol (80 mL), concentrated hydrochloric acid (8 mL) was added to the solution, and heated under reflux for 1 hr in the dark. After the reaction was completed, the reaction solution was concentrated under reduced pressure, extracted with ethyl acetate and washed with water, dried and concentrated, and purified by column chromatography (dichloromethane / methanol=60 / 1) to obtain an orange solid 3-5 (3.3 g, 83%). 1 H NMR (400MHz, CD3OD) δ7.57(d,J=9.0Hz,1H),7.23(s,1H),6.89–6.85(m,1H),6.85(d,J=1.3Hz,1H),4.77(d,J=1.4Hz,2H). 13 C NMR(101MHz,CD3OD)δ199.6,163.2,159.3,149.4,126.3,122.7,115.6,113.0,103.3,60.5.HRMS calcd for C 10 H8O3S[M+H] + :209.0267; found:209.0267.

[0061] Compound F and compound G Synthesis

[0062] 3-5 (1.8 g, 8.6 mmol) was dissolved in tetrahydrofuran (30 mL). When the solution was cooled to 0°C, triethylamine (1.8 mL, 13.0 mmol) and tetrahydrofuran solution (20 mL) of p-nitrophenyl chloroformate (2.6 g, 13.0 mmol) were added in sequence. The reaction solution was heated to room temperature and stirred in the dark for 4 hours. After the reaction was completed, the reaction solution was washed with saturated sodium bicarbonate solution, dried and concentrated to obtain a crude product 3-6 (1.4 g, 43%), which can be directly used in the next step. Subsequently, N α-tert-Butyloxycarbonyl-L-lysine (989 mg, 4.0 mmol) was dissolved in N, N-dimethylformamide (10 mL), and triethylamine (186 μL, 1.3 mmol) and N, N-dimethylformamide (10 mL) solution in which 3-6 (1 g, 2.7 mmol) was dissolved were added to the solution in sequence. The reaction solution was stirred overnight at room temperature in the dark. After the reaction was completed, the reaction solution was concentrated under reduced pressure, the remaining reaction solution was dissolved in ethyl acetate, and 0.1 M hydrochloric acid solution was added for acidification, the organic phase was collected, dried and concentrated, and purified by column chromatography (dichloromethane / methanol = 20 / 1) to obtain an orange oily liquid 3-7 (566 mg, 44%). 1 H NMR (400MHz, CD3OD) δ7.55(d,J=8.7Hz,1H),7.06(s,1H),6.86(dd,J=8.7,2.4Hz,1H),6.82(d,J=2 .4Hz,1H),5.23(s,2H),4.11–4.00(m,1H),3.15(t,J=6.6Hz,2H),1.85–1.63(m,2H),1.42(s,13H). 13 C NMR(101MHz,CD3OD)δ198.9,163.4,159.3,157.8,144.6,126.4,123.2,115.7, 112.6,103.4,80.5,62.4,54.8,49.4,41.6,32.5,30.9,30.3,28.7,24.1.HRMS calcd for C 22 H 28 N2O8S[M+H] + :479.1494; found:479.1495.

[0063] S K

[0064] Synthesis

[0065] 3-7 (566 mg, 1.2 mmol) was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (5 mL) was subsequently added. The reaction solution was stirred at room temperature in the dark for 1 hr. After the reaction was completed, the reaction solution was concentrated under reduced pressure. The solid obtained after concentration was dissolved in methanol and added dropwise to ether to precipitate. The precipitate was collected and recrystallized again, and filtered and dried to obtain an orange solid SCouK (354 mg, 79%). 1H NMR (400MHz, CD3OD) δ7.60(d,J=8.7Hz,1H),7.08(s,1H),6.89(dd,J=8.7,2.1Hz,2H),6.86(d,J =2.3Hz,1H),5.26(s,2H),3.78(s,1H),3.22–3.13(m,2H),1.99–1.85(m,2H),1.64–1.47(m,4H). 13 C NMR (101MHz,d 6 -DMSO)δ196.4,170.3,162.8,157.7,155.3,144.7,125.9,121.0,115.2,110.6,102.2,60.5,53.9,40.3,30.6,29.0,22.3.HRMS calcd for C 17 H 20 N2O6S[MH] - :379.0969; found:379.0972.

[0066] Example 2: Photo-uncaging characterization experiment of SCouK

[0067] In order to test whether SCouK can undergo photo-uncaging reaction, this chapter synthesized 9-fluorenylmethoxycarbonyl (Fmoc) protected SCouK, namely Fmoc-SCouK ( Figure 4 A). The synthesis of Fmoc-SCouK is similar to that of compound G (3-7) in the above experiment, and will not be repeated here. Fmoc-SCouK and SCouK have the same thiocoumarin photocage protecting group, but its expected photolysis product FmocK ( Figure 4 A) It has strong absorption at 254nm, which is convenient for HPLC detection, so this study selected Fmoc-SCouK as a model molecule. In order to simulate physiological conditions, this study selected PBS buffer as the solvent for the photo-uncaging reaction of Fmoc-SCouK. Considering that 530nm green light has the advantages of long wavelength, low phototoxicity and penetration compared with other light sources, it is an ideal light source for photo-uncaging. Therefore, this study tried to use 530nm green light as the light source to trigger the photo-uncaging reaction of Fmoc-SCouK. Specifically, this study irradiated the PBS solution of Fmoc-SCouK with 530nm light for different times, and then performed HPLC-MS analysis.

[0068] like Figure 4 The analysis results of B showed that the chromatographic peak corresponding to Fmoc-SCouK decreased with the extension of illumination time, and a new chromatographic peak gradually appeared at the retention time of 10.8min. Mass spectrometry analysis showed that the chromatographic peak was FmocK ( Figure 4 A). For another expected reaction product, SCou-OH, due to weak absorption at 254nm, the generation and enhancement of the corresponding chromatographic peak of SCou-OH was not clearly observed on the HPLC chromatogram (detector wavelength was 254nm). By searching for the molecular weight corresponding to SCou-OH, this study found that the product appeared at a retention time of 9.2min, and the corresponding positive ion molecular weight was 209.0267, which was consistent with the theoretical molecular weight ( Figure 4 C), proving that SCou-OH is produced by photodecage of Fmoc-SCouK. Therefore, these results indicate that Fmoc-SCouK does undergo decage reaction to generate FmocK and SCou-OH after green light illumination, releasing the protected amino group, which is consistent with expectations. Figure 4 The chromatographic peak areas in B were used for relative quantification and conversion analysis of Fmoc-SCouK and FmocK. Figure 4 D The results showed that Fmoc-SCouK had reacted 79% at 30 seconds of illumination and was almost completely photolyzed at 2 minutes, with its half-time of photo-uncaging being about t1 / 2 = 12.8 seconds. Correspondingly, FmocK was generated in large quantities after 30 seconds of illumination, and was almost completely released at 2 minutes of illumination. In summary, the HPLC-MS analysis results were consistent with the expectations of this study, confirming that thiocoumarin can undergo photo-uncaging reaction after green light irradiation within 2 minutes, which laid the foundation for its application in the precise regulation of protein activity.

[0069] Example 3: SCouK site-specific protein introduction in prokaryotic systems

[0070] 1) Screening of pyrrolysyl-tRNA synthetase mutants that recognize SCouK

[0071] In order to obtain an active site mutant of pyrrolysyl-tRNA synthetase synthetase (MmPylRS) capable of inserting SCouK, the present invention first screened a synthetase mutant library.

[0072] SCouK computationally modeled into the pyrrolysine binding pocket of pyrrolysyl-tRNA synthetase, and noted that SCouK has a coumarin-based side chain that may sterically clash with many residues around the pocket (e.g., 306A, 309G, 348F, 405R, and 413V). Therefore, by rationally designing these residues to sterically less hindered amino acids, a series of active site mutants based on wild-type pyrrolysyl-tRNA synthetase were constructed, as shown in the following table.

[0073] Mutation Group Mutation site Group 1 111D / 306G / 309A / 384F Group 2 309A / 348S / 384F Group 3 309A / 348F / 384F Group 4 309A / 348F Group 5 306A / 348F Group 6 306G / 309G / 384F Group 7 306G / 309A / 384F Group 8 306A / 309G / 384F Group 9 306A / 309A / 384F / IPYE Group 10 306A / 309A / 413V / 384F Group 11 306A / 309A / 405R / 384F Group 12 306A / 309A / 350A / 384F Group 13 306A / 309A / 348S / 405R / 384F Group 14 306A / 309A / 348S / 384F Group 15 306A / 309A / 348A / 384F Group 16 306A / 309A / 384F Group 17 384F

[0074] In order to test the recognition ability of these synthetase mutants to SCouK, this study used EGFP-Y39TAG as a model protein and evaluated the recognition efficiency of synthetase mutants to SCouK by detecting the fluorescence intensity of EGFP-Y39SCouK expressed in E. coli. The screening results showed that various MmPylRS mutants could recognize SCouK and insert it site-specifically into EGFP, such as Figure 5 As shown in A, MmPylRS-306A / 309A / 405R / 384F I has the highest recognition efficiency for SCouK. Therefore, this synthetase mutant, hereinafter referred to as SCouKRS, was used in subsequent experiments in this study.

[0075] 2) Use SCouK to insert target proteins into Escherichia coli.

[0076] Subsequently, in order to investigate the site-specific insertion efficiency of SCouK in the target protein of E. coli, this study used sfGFP as a model protein, co-transfected the plasmids containing SCouKRS and sfGFP-N150TAG into the E. coli BL21 (DE3) strain, selected single clones, amplified the strains, and induced sfGFP expression to site-specifically insert SCouK. Figure 5 As shown in B, immunoblotting analysis verified the normal expression of sfGFP-N150SCouK full-length protein. Figure 5 The results of immunoblot analysis of C showed that SCouK had a high insertion efficiency even at a low concentration of 0.25 mM.

[0077] 3) Verify the fidelity of SCouK insertion into E. coli

[0078] The sfGFP-N150SCouK protein recombinantly expressed in E. coli was affinity purified by Ni-NTA resin and analyzed by mass spectrometry. Figure 6 As shown in A, mass spectrometry analysis showed that the molecular weight of the recombinant protein was 28075.30 Da, which was basically consistent with the theoretical calculated value of 28075.28 Da, indicating that SCouK was site-specifically inserted into the target protein by SCouKRS in Escherichia coli in an efficient and high-fidelity manner.

[0079] The sfGFP-N150SCouK protein prepared above was used to further verify the photo-uncaging reaction of SCouK at the protein level, that is, the sfGFP-N150SCouK protein was irradiated with green light for 1 min and then subjected to mass spectrometry analysis. Figure 6As shown in Figure B, mass spectrometry analysis did not find the molecular weight corresponding to sfGFP-N150SCouK, but showed that the molecular weight of the protein after illumination was 27840.56Da, which exactly matches the theoretical molecular weight of sfGFP-N150K protein, 27841.39Da. This result shows that sfGFP-N150SCouK can release the lysine residue at position 150 after green light uncaging and convert it into sfGFP-N150K, verifying the feasibility of SCouK light uncaging reaction at the protein level.

[0080] 4) Use SCouK to insert target proteins into mammalian cells.

[0081] Specifically, in this study, HEK293T cells were co-transfected with plasmids encoding sfGFP-N150TAG and SCouKRS. After overnight culture, the cells were collected and lysed. The lysate was analyzed by SDS-PAGE and immunoblotting to verify that SCouK was site-specifically inserted into the sfGFP model protein, resulting in normal full-length expression of the protein, such as Figure 7 As shown in A. Subsequently, this study optimized the SCouK concentration. Figure 7 As shown in B, immunoblot analysis showed that the expression of sfGFP-N150SCouK protein was SCouK concentration-dependent. When the SCouK concentration reached 200 μM, sfGFP could be introduced with high efficiency. At the same time, the green fluorescence of sfGFP incorporated with SCouK was also observed through fluorescence imaging experiments, and the green fluorescence was stronger at 200 μM, that is, the expression level of sfGFP was high, as shown in Figure 7 C. After quantitative analysis of cell fluorescence images, this study found that even at a concentration as low as 200 μM, SCouK could still insert sfGFP at a high efficiency, as shown in Figure 7 As shown in D.

[0082] The above results indicate that the thiocoumarin-based unnatural amino acid SCouK can be efficiently site-specifically introduced into Escherichia coli and mammalian cells through genetic codon expansion technology.

[0083] Example 4: SCouK for fluorescence signal detection of photoactivated fluorescent protein

[0084] To verify that SCouK can be used for site-specific regulation of protein activity, this study first selected EGFP as the target protein for light activation.

[0085] EGFP has the advantages of high fluorescence intensity, strong stability, and good cell compatibility, and is one of the most widely used fluorescent proteins. This study envisions introducing SCouK into the key lysine site of EGFP, using light to cause SCouK to undergo a rapid photo-uncaging reaction, promote the folding and maturation of the EGFP chromophore, and thus achieve precise activation of the fluorescence signal. K85 is a key lysine site of EGFP, which can form hydrogen bonds or salt bridges with important residues (C70, S72, D82) that control the formation and oxidation of the chromophore. Therefore, mutations at the K85 site can effectively inhibit the maturation of the EGFP chromophore and the fluorescence signal. In addition, the introduction of photocaged unnatural amino acids at the K85 site of EGFP can also inhibit the maturation of its chromophore, resulting in the disappearance of EGFP fluorescence; while the photocaged unnatural amino acids at the 85th position can generate lysine residues in situ after illumination, restoring the EGFP fluorescence. Based on this, this study intends to insert SCouK into the K85 site of EGFP, express EGFP-K85SCouK protein in mammalian cells, and use green light to mediate the photo-uncaging reaction, so as to evaluate the effect of EGFP photoactivation by detecting the changes in EGFP fluorescence signal.

[0086] In order to achieve visual tracking of EGFP-K85SCouK protein and relative quantitative analysis of EGFP fluorescence signal, this study selected EGFP-K85TAG-mCherry fusion protein developed by our laboratory as the site-directed introduction protein of SCouK. Figure 8 As shown. Compared with EGFP-K85TAG, the difference between EGFP-K85TAG-mCherry is that the red fluorescent protein mCherry is fused to the C-terminus of EGFP. Once SCouK is inserted into EGFP, the fusion protein EGFP-K85TAG-mCherry will be fully expressed. The fluorescence intensity of mCherry is only related to the insertion efficiency of SCouK, and has nothing to do with whether SCouK undergoes photo-uncaging reaction. Therefore, mCherry can be used as a fluorescent tag to visually track the distribution and expression of EGFP-K85SCouK in cells, and can also be used as an internal reference protein to perform relative quantitative analysis of EGFP photoactivation through the fluorescence ratio between EGFP / mCherry, eliminating the interference of factors such as protein expression level and cell state, and improving the accuracy of detection.

[0087] Therefore, this study co-transfected HEK293T cells with plasmids encoding EGFP-K85TAG-mCherry and SCouKRS, and added 200μM ScouK to the cell culture medium to express the target protein EGFP-K85SCouK-mCherry. Then, different wavelengths of light were used for illumination, and the optimal illumination wavelength was selected by cell fluorescence imaging. This study also explored different illumination times at this wavelength to obtain the optimal experimental conditions for light activation.

[0088] Fig. 9 A The results showed that the red fluorescence of mCherry could be observed in the cells under all conditions, proving the successful expression of EGFP-K85SCouK-mCherry. Only after irradiation with a 365nm, 405nm, 460nm or 530nm light source for 1 minute, can the green fluorescence from EGFP be observed in the cells, and it coincides with the mCherry signal. Under dark and light-free conditions, no obvious green fluorescence was observed in the cells, but the expression and distribution of EGFP-K85SCouK in the cells could be observed through the mCherry red signal, indicating that EGFP-K85SCouK-mCherry does not undergo photo-uncaging reaction under dark conditions. It is worth mentioning that no green fluorescence was observed in the cells under 650nm light conditions, indicating that irradiation with this wavelength cannot induce photo-uncaging reaction. Subsequently, this study used ImageJ software to perform relative quantitative analysis of the fluorescence intensity of the fluorescence images, and used the relative fluorescence intensity ratio of EGFP to mCherry (EGFP / mCherry) as the standard for evaluating the photoactivation efficiency of EGFP. Fig. 9 As shown in B, the EGFP / mCherry fluorescence intensity ratio under 365nm, 405nm, 460nm, and 530nm illumination conditions increased by 9.2, 8.9, 10.1, and 11.0 times, respectively, compared with that under dark conditions. These results verify that SCouK can be site-specifically inserted into the key K85 site of EGFP in mammalian cells, effectively "turning off" the EGFP fluorescence signal in a dark environment; after SCouK photo-uncaging is induced by 365nm, 405nm, 460nm, or 530nm light source irradiation, the EGFP chromophore folds and matures, thereby "turning on" green fluorescence. In addition, these results also indicate that 530nm green light is the preferred activation wavelength for SCouK photo-uncaging.

[0089] After determining that 530 nm is the optimal wavelength for photo-uncaging of SCouK, this study continued to explore the effect of illumination time on photo-uncaging efficiency under 530 nm illumination conditions. Specifically, this study treated HEK293T cells expressing EGFP-K85SCouK-mCherry with illumination for 10 s, 30 s, 60 s, 120 s, and 300 s, respectively, and then performed fluorescence imaging analysis. Fig.10A The results showed that after illumination, green fluorescence of EGFP was produced in the cells, and red fluorescence of co-localized mCherry was also observed. However, cells in the dark and without illumination had almost no green fluorescence signal, and only mCherry fluorescence signal, proving the successful expression and light activation of EGFP-K85SCouK-mCherry. Subsequently, this study conducted a quantitative analysis of the fluorescence intensity of the fluorescence images and calculated the relative fluorescence intensity ratio of EGFP / mCherry for each group of images. Fig.10 As shown in B, the EGFP / mCherry fluorescence intensity ratio under 10s, 30s, 60s, 120s and 300s illumination conditions increased by 6.2, 7.5, 7.1, 7.1, 6.9 and 5.3 times respectively compared with that under dark conditions. Fig. 9 There is a big difference in the growth multiples shown in B, which may be due to the differences in experimental conditions such as the expression time of the target protein and the instrument parameters used in the two experiments. But in summary, these results once again verified that the green fluorescence signal of EGFP-K85TAG-mCherry protein can be activated by light control in mammalian cells through site-specific introduction of SCouK, and also showed that SCouK can achieve rapid light uncaging reaction and regulate protein activity on a time scale of seconds.

[0090] Example 5: SCouK is used for light-activated luciferase activity experiments

[0091] To test whether the photo-uncaging reaction of SCouK can be used to activate enzymes in living cells, firefly luciferase (fLuc) was selected as the target protein in this example.

[0092] In the presence of adenosine triphosphate (ATP), oxygen and magnesium ions, fLuc catalyzes the decarboxylation and oxidation of luciferin substrate (D-Luciferin) to generate oxyluciferin (Oxyluciferin), while emitting bioluminescence at 540–600 nm. Fig.11 As shown in the figure. The catalytic activity of fLuc depends on multiple key amino acid residues, among which lysine at position 529 (K529) can generate electrostatic interactions with the carboxylic acid group of the fluorescein substrate and the phosphate group of ATP, helping the substrate to maintain the correct reaction conformation and playing an important role in stabilizing the transition state formed during the adenylation process of the substrate; when the K529 site mutates, the enzyme activity of fLuc is significantly reduced or even inactivated, indicating that K529 has a significant effect on the catalytic activity of fLuc.

[0093] The genetic codon expansion technique was used to introduce SCouK into the K529 site of fLuc, inhibiting its enzymatic activity. Subsequently, the lysine residue at the 529 site was released by light, restoring the activity of fLuc (see Fig.11 In addition, in order to compare the photo-uncaging efficiency of SCouK with other photo-caged unnatural amino acids, the reported photo-caged unnatural amino acid HCK was selected as a control. Fig.12 As shown in A, the coumarin carbonyl oxygen is not replaced, and a series of target proteins can be activated by ultraviolet light-mediated photo-uncaging reaction.

[0094] Specifically, this study co-transfected HEK293T cells with a His-tagged fLuc-K529TAG plasmid and a MmPylRS mutant plasmid corresponding to UAA, and expressed fLuc-K529BocK, fLuc-K529HCK and fLuc-K529SCouK proteins in the presence of BocK, HCK and SCouK, respectively. The cells were then irradiated with light sources of different wavelengths for 1 min, and then the luciferin substrate reagent was added. The bioluminescent signal was detected and quantitatively analyzed using a microplate reader to evaluate the changes in fLuc enzyme activity. Fig.12 B The results showed that, as expected, after irradiation with 365nm, 405nm, 460nm, and 530nm light sources, the catalytic activity of fLuc-K529SCouK was restored, resulting in a significant enhancement of the bioluminescent signal compared to dark conditions and 630nm light conditions; at the same time, 365nm and 405nm light also led to a significant enhancement of the catalytic activity and bioluminescent signal of fLuc-K529HCK compared to dark conditions; as a negative control, fLuc-K529BocK, which is insensitive to light, did not show obvious changes in bioluminescent signal intensity before and after illumination. After 365nm and 405nm light, the absolute intensity of the bioluminescent signal catalyzed by fLuc-K529SCouK was 4.4 and 3.3 times that of fLuc-K529HCK under the corresponding conditions, respectively, indicating that SCouK has a higher detachment efficiency than HCK. At the same time, Fig.12 C Western blot analysis results showed that the expression level of fLuc-K529HCK in cells was slightly higher than that of Luc-K529SCouK, which also verified that under the same illumination conditions, fLuc-K529SCouK had higher enzyme catalytic activity than fLuc-K529HCK because SCouK had a higher uncaging efficiency, rather than because fLuc-K529SCouK protein was expressed more. In addition, fLuc-K529HCK can only be activated by ultraviolet light sources (365nm and 405nm), and cannot be uncaged by long-wavelength light such as 460nm and 530nm, showing certain limitations.

[0095] like Fig.12 As shown in D, living cells expressing fLuc-K529SCouK were irradiated with light of different wavelengths for bioluminescence visualization detection by gel imaging. The results showed that under 365nm, 405nm, 460nm, and 530nm illumination conditions, cells expressing fLuc-K529SCouK showed significant bioluminescence signals, while under dark conditions or after 630nm illumination, almost no bioluminescence signals were observed in cells expressing fLuc-K529SCouK.

[0096] In summary, these results prove that the introduction of SCouK at the K529 site can effectively inhibit the enzymatic activity of fLuc, and 530nm green light irradiation can quickly restore the enzymatic activity of fLuc-K529SCouK, indicating that SCouK can be used for precise activity regulation of fLuc through its own photocage. More importantly, compared with the known photocaged unnatural amino acid HCK, SCouK has a longer uncage wavelength and faster and higher uncage efficiency.

[0097] Example 6: SCouK for light activation of MEK1 and Src kinases

[0098] like Fig.13 As shown in A, when the photocaged non-natural amino acid is introduced into the key lysine site K97 of MEK1, it occupies the kinase ATP binding pocket, inhibiting the activity of MEK1 kinase. When light uncaging reaction is triggered by light, the free lysine residue is released, and the activity of MEK1 kinase is restored, which then phosphorylates the downstream protein ERK1 / 2 and causes ERK1 / 2 to enter the nucleus, triggering downstream signal transduction.

[0099] like Fig.13As shown in B, SCouK can be inserted into the K97 site of MEK1-ΔN through genetic codon expansion technology, and has a high insertion efficiency. The phosphorylation level of ERK1 / 2 is low under dark conditions, indicating that the activity of MEK1-ΔN-K97SCouK is effectively inhibited and cannot be phosphorylated. As the illumination time gradually increases, the phosphorylation level of ERK1 / 2 gradually increases and reaches the highest level under 120s illumination conditions, indicating that illumination can indeed cause MEK1-ΔN-K97SCouK to undergo photo-uncaging, release key lysine residues, and thus restore its kinase activity. At the same time, this study used BocK as a negative control non-natural amino acid to express MEK1-ΔN-K97BocK in cells. Immunoblotting analysis showed that MEK1-ΔN-K97BocK could not cause an increase in ERK phosphorylation signals under conditions of illumination or not, which further demonstrated that the loss of kinase activity of MEK1-ΔN-K97TAG was caused by the introduction of non-natural amino acids at the K97 site. These results indicate that by specifically introducing SCouK at the K97 site of MEK1-ΔN, the activity of MEK1-ΔN-K97SCouK can be activated by short-term illumination, thereby achieving precise regulation of downstream signaling pathways.

[0100] like Fig.14 As shown in A, plasmids encoding Src-Y527F-K295TAG and SCouKRS were transfected into HEK293T cells to express Src-Y527F-K295SCouK containing an HA tag. The cells were then illuminated with green light, and the phosphorylation of Src's own Y416 site and the phosphorylation levels of its downstream substrate proteins were detected by immunoblotting analysis.

[0101] like Fig.14As shown in Figure B, SCouK can be introduced into the 295 site of Src-Y527F with high efficiency, and Src-Y527F-K295SCouK protein is expressed in HEK293T cells. As a positive control, the active mutant of Src-Y527F showed a strong Y416 autophosphorylation level (Src-pY416) after expression in the cells, and significantly enhanced the tyrosine phosphorylation level of the downstream substrate protein (p-Tyr). Under dark conditions, Src-Y527F-K295SCouK only showed weak autophosphorylation, and the overall tyrosine phosphorylation signal in the cell was also weak, indicating that the Src-Y527F-K295SCouK inserted into SCouK was in an inactive state. As the illumination time gradually increased, the autophosphorylation level of Src-Y527F and the overall tyrosine phosphorylation level of the downstream substrate protein gradually increased, and reached saturation at 60s of illumination. This indicates that Src-Y527F-K295SCouK can regenerate the native lysine residue at the K295 site through green light-mediated SCouK photouncaging reaction, thereby restoring its kinase activity.

[0102] The above results indicate that SCouK can be used for rapid and precise activity regulation of MEK1 and Src kinases in living cells, and has a certain universality in different kinase families.

[0103] The present invention is further described by the accompanying drawings and the following examples, which are only for illustrating specific embodiments of the present invention and should not be construed as limiting the scope of the present invention in any way.

[0104] In summary, the present invention successfully developed a non-natural amino acid SCouK based on thiocoumarin, and used genetic codon expansion technology to site-specifically insert it into the key lysine site of the target protein, making it inactive; then, the thiocoumarin group was removed by green light-mediated photo-decaging reaction, and natural lysine was regenerated to restore the activity of the protein, thereby realizing the function and activity regulation of multiple target proteins in living cells.

[0105] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art may make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should all fall within the scope of the claims attached to the present invention.

Claims

1. A class of unnatural amino acids, characterized in that The non-natural amino acid is a photocaged non-natural amino acid based on thiocoumarin, and its structural formula is shown below: Specifically, its structure contains lysine residues as well as thiocoumarins.

2. The method for preparing the non-natural amino acid according to claim 1, characterized in that: The steps include: Step 1) using resorcinol as a starting material, condensing and cyclizing with ethyl 4-chloroacetoacetate under acidic conditions to obtain compound A (3-1); Step 2) hydrolyzing compound A to produce compound B (3-2); Step 3) Protecting the dihydroxyl group of compound B (3-2) with acetyl group to obtain compound C (3-3); Step 4) The obtained compound C (3-3) is subjected to a thiolation reaction in the dark to generate compound D (3-4); Step 5) removing the acetyl group from the obtained compound D (3-4) with hydrochloric acid to obtain compound E (3-5); Step 6) Compound E (3-5) is reacted with p-nitrophenyl chloroformate to obtain an activated ester compound F (3-6) of thiocoumarin; Step 7) The obtained activated ester compound F (3-6) is connected with Nα-tert-butyloxycarbonyl-L-lysine to generate compound G (3-7); Step 8) Use trifluoroacetic acid to remove the tert-butyloxycarbonyl group to obtain a photocaged unnatural amino acid.

3. A protein expression system, a translation system and a cell comprising the unnatural amino acid of claim 1.

4. Use of the non-natural amino acid as claimed in claim 1 for site-specific introduction into a target protein in a prokaryotic or eukaryotic system to regulate its activity.

5. Use of the non-natural amino acid as claimed in claim 1 in enhancing the fluorescence signal activation of green fluorescent protein.

6. Use of the non-natural amino acid as claimed in claim 1 in protein kinase activity.

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