CN118420494B - Hydroxyl radical reactive unnatural amino acid and preparation method and application thereof - Google Patents

Abstract

The present invention relates to a hydroxyl radical reactive non-natural amino acid. Specifically, the present invention discloses a reactive non-natural amino acid HBCF that can be used to construct a genetically encoded hydroxyl radical fluorescent probe, a preparation method, and its application in the detection and imaging of hydroxyl radicals. The important significance of the present invention lies in the site-specific introduction of HBCF into the key tyrosine site of the fluorescent protein, and the recombinant expression and purification in Escherichia coli to prepare the fluorescent probe cpsfGFP-Y163HBCF. The probe is applied to the bacterial level, and it is proved by immunoblot analysis and fluorescence imaging that HBCF can be site-specifically inserted into the target protein in bacteria, and it is preliminarily proved that the probe cpsfGFP-Y163HBCF can perform fluorescent detection of hydroxyl radicals.

Description

Hydroxyl radical reactive unnatural amino acid and preparation method and application thereof

Technical Field

The invention relates to the field of biological sample detection, in particular to the field of detection of OH in biological samples.

Technical Field

Reactive oxygen species (Reactive oxygen species, ROS) play an important regulatory role in many physiological and pathological processes. Prior studies have shown that oxidative stress in cells is caused by an imbalance between the production and elimination of reactive oxygen species in cells. OH (hydroxyl radical) is the most reactive intracellular ROS, and when its concentration is abnormally increased, it attacks biological macromolecules in the body, causing serious cell damage, and further inducing various diseases including cerebral apoplexy, parkinson, alzheimer, rheumatoid arthritis, cardiovascular diseases and the like. Thus, there is a need for a highly selective and sensitive method for real-time detection of the generation, distribution and dynamic fluctuations of OH in the life system to fully understand its biological role. However, real-time detection of endogenous OH is extremely challenging due to the high reactivity of endogenous OH, short lifetime, and low intracellular concentration. With the development of genetic codon extension techniques, scientists developed a series of unnatural amino acids (Unnatural amino acid, UAA) that are capable of reacting with biologically active species for the construction of chemically reactive genetically encoded fluorescent probes, and have successfully achieved detection and imaging of ONOO ^-, etc.

The group of the task of Liu Zhibo at Beijing university in 2020 developed a fluorescent probe DHBC-MeRho with 3, 5-dihydroxybenzyloxycarbonyl (3, 5-Dihydroxybenzyl carbamate, DHBC) as a masking group, the DHBC group of which was selectively removed by nuclear radiation generated OH, which reacted rapidly, releasing fluorescence.

Based on the above, the invention researches the chemical reaction of the DHBC group and the OH and the genetic code expanding technology, and develops and synthesizes the OH reactive unnatural amino acid HBCF for preparing the chemically reactive genetic code OH probe.

Disclosure of Invention

The invention aims to provide an unnatural amino acid HBCF (tyrosine derivative) capable of reacting with OH and a method for constructing a chemically reactive genetic code OH probe by using the same. As shown in FIG. 1, the research of the invention designs and synthesizes a tyrosine derivative 4- (3, 5-dihydroxybenzyloxycarbonylamino) -L-phenylalanine (4- (3, 5-Dihydroxybenzyloxycarbonylamino) -L-phenylalanine, HBCF) containing a DHBC side chain, wherein the DHBC side chain group can selectively react with OH to generate a tyrosine analogue 4-Amino-L-phenylalanine (4-Amino-L-phenylalanine, AF) in situ after ortho-hydroxylation, 1, 4-or 1, 6-elimination. The invention researches and further utilizes the genetic code expansion technology to insert HBCF site specificity into key sites of the chromophore of the annular arrangement super-folding green fluorescent protein (circularlypermutated superfolded green fluorescent protein, cpsfGFP) to construct a genetic code OH fluorescent probe. As shown in FIG. 2, since the key site of the chromophore is masked by the side chain of HBCF and introduced into cpsfGFP of HBCF, fluorescence is hardly generated before the reaction with OH, and after the reaction with OH, the DHBC group of HBCF is cleaved to generate an analogue AF of tyrosine, so that cpsfGFP fluorescence is obviously enhanced, thereby realizing fluorescence detection of OH.

In order to achieve the above purpose, the present invention provides the following technical solutions:

The invention provides a non-natural amino acid lysine analogue capable of reacting with OH, which has the following structural formula:

Characterized by comprising catechol and 4-amino-L-phenylalanine structures.

Specifically, the unnatural Amino acid is a tyrosine derivative 4- (3, 5-dihydroxybenzyloxycarbonylamino) -L-phenylalanine (4- (3, 5-Dihydroxybenzyloxycarbonylamino) -L-phenylalanine, HBCF) containing a DHBC side chain, wherein the DHBC side chain group can selectively react with OH to generate a tyrosine analogue 4-Amino-L-phenylalanine (4-Amino-L-phenylalanine, AF) in situ after ortho-hydroxylation, 1, 4-or 1, 6-elimination.

The invention also provides a chemical synthesis method of the unnatural amino acid HBCF, which comprises the following steps:

step 1) protecting the carboxylic acid group of compound 1 (nα -t-butoxycarbonyl-4-amino-L-phenylalanine) with methyl ester to give compound 2;

Step 2), protecting hydroxyl of a compound 3 (3, 5-dihydroxybenzoic acid methyl ester) by tert-butyldimethyl chlorosilane to obtain a compound 4;

step 3) reducing the compound 4 by lithium aluminum hydride to obtain a compound 5;

step 4) protecting the amino group of compound 4 with a t-butoxycarbonyl group to give compound 5;

Step 5) reacting the compound 5 with triphosgene and then reacting with the compound 2 to generate a compound 6;

And 6) sequentially deprotecting tetrabutylammonium fluoride, lithium hydroxide and trifluoroacetic acid to obtain the hydroxyl radical reactive unnatural amino acid HBCF.

The invention also provides a compound for connecting any one of the above unnatural amino acids.

The invention also provides a composition comprising any of the unnatural amino acids described above.

The invention also provides an expression system or a translation system comprising any of the unnatural amino acids described above.

The application of the hydroxyl radical reactive unnatural amino acid in constructing a hydroxyl radical reactive fluorescent probe;

The hydroxyl radical reactive fluorescent probe is a genetically encoded fluorescent protein probe which introduces any one of the unnatural amino acids at a specific site of a biomacromolecule, and preferably the probe is cpsfGFP-Y163 HBCF.

The hydroxyl radical reactive fluorescent probe comprises the composition of any one of the hydroxyl radical reactive fluorescent probes.

The hydroxyl radical reactive fluorescent probe comprises an expression system or a translation system of any one of the hydroxyl radical reactive fluorescent probes.

The invention also provides a method for detecting hydroxyl radical reactivity in a sample, which comprises the step of imaging or detecting hydroxyl radical reactivity in the sample by using any one of the hydroxyl radical reactive fluorescent probes, the composition or the expression system or the translation system.

Compared with the prior art, the invention has the beneficial effects that:

1) The invention develops a novel OH-reactive UAA based on tyrosine analogues and OH reactive groups, namely HBCF by utilizing a genetic code expansion technology, and develops a novel genetic code OH fluorescent probe cpsfGFP-Y163HBCF based on the novel OH-reactive UAA, so that the selective fluorescence detection of OH can be carried out in vitro;

2) The invention synthesizes the unnatural amino acid HBCF based on the OH reactive group DHBC and 4-amino-phenylalanine AF, and verifies that the HBCF can react with OH to generate AF by utilizing LC-MS. According to the research, a series of MmPylRS mutants are screened to obtain a synthetase mutant (HBCF-RS) capable of effectively recognizing HBCF, and the site-specific insertion of the HBCF into target protein in escherichia coli of a prokaryotic system is realized;

3) The invention utilizes the genetic code expansion technology to introduce HBCF into the key tyrosine locus of fluorescent protein at fixed point, recombining and expressing in colibacillus and purifying to prepare fluorescent probe cpsfGFP-Y163HBCF, carrying out systematic characterization on response time, sensitivity, selectivity and the like of the probe for detecting OH in vitro by fluorescence spectrum analysis and fluorescence intensity quantification, and further proving that the HBCF on the probe is enhanced by fluorescence caused by tyrosine analogue AF generated by reaction with OH by mass spectrometry and negative control experiments.

Drawings

FIG. 1 Structure of HBCF and its selective reaction with OH;

FIG. 2 synthetic route pattern of HBCF;

FIG. 3 reaction of Boc-HBCF with OH;

FIG. 4 LC-MS results after reaction of Boc-HBCF with OH;

FIG. 5 is a graph showing the results of screening for a synthase mutant recognizing HBCF;

FIG. 6 shows the results of site-directed insertion analysis of HBCF in E.coli (A) by immunoblotting analysis, the site-directed insertion efficiency of two synthetases on HBCF was compared, the concentration of HBCF was 1mM, (B) the chemical structure of BocK, (C) by immunoblotting analysis, the protein expression of sfGFP-N150BocK and sfGFP-N150HBCF was compared, the concentration of BocK was 1mM, the concentration of HBCF was 2mM, and (D) by immunoblotting analysis and (E) by fluorescence intensity analysis, the expression of sfGFP-N150HBCF protein was analyzed under different concentrations of HBCF. CB-Coomassie brilliant blue staining.

FIG. 7 purification and characterization of sfGFP-N150HBCF protein in E.coli (A) SDS-PAGE electrophoresis of purified sfGFP-N150HBCF protein (B) ESI-MS mass spectrometry of sfGFP-N150HBCF protein, mass spectra were deconvolved with BioPharma Finder. CB-Coomassie brilliant blue staining.

FIG. 8 shows the expression of several fluorescent proteins inserted into HBCF in E.coli (A) immunoblotting analysis of EGFP-Y66TAG, sfGFP-Y66TAG, cpsfGFP-Y163TAG and mCherry-Y72TAG inserted into HBCF at a concentration of 2mM, and (B) immunoblotting analysis of cpsfGFP-Y163TAG protein expression at different concentrations of HBCF. CB-Coomassie brilliant blue staining.

FIG. 9 SDS-PAGE and mass spectrometry of cpsfGFP-Y163HBCF probe (A) SDS-PAGE electrophoresis of purified cpsfGFP-Y163HBCF protein and (B) ESI-MS mass spectrometry of cpsfGFP-Y163HBCF protein, mass deconvolved with BioPharma Finder. CB-Coomassie brilliant blue staining, "-Met" indicates that the N-terminal initiator methionine is cleaved by methionine aminopeptidase in E.coli.

FIG. 10 fluorescence response of cpsfGFP-Y163HBCF probe to OH in vitro;

FIG. 11 characterization of cpsfGFP-Y163HBCF probe for in vitro detection of OH. (a) linear relationship between fluorescence intensity at 510nm and concentration of OH for 0.5 μm probe (t=60 min), (B) fluorescence response at 510nm for low concentration of OH for 0.5 μm probe (t=60 min), statistically analyzed by one-way ANOVA with p <0.01, (C) kinetics of fluorescence response for 0.5 μm probe to OH (4 μm);

FIG. 12 selectivity of cpsfGFP-Y163HBCF probe for in vitro detection of OH;

FIG. 13 is a mass spectrometry chart of ESI-MS after cpsfGFP-Y163HBCF has reacted with OH;

FIG. 14 synthetic route pattern of unnatural amino acid BCF;

FIG. 15 is a screening chart of synthetase mutants recognizing BCF;

FIG. 16 shows the site-directed insertion protein analysis of BCF in E.coli (A) immunoblotting analysis compares the protein expression of sfGFP-N150BocK and sfGFP-N150BCF with BocK concentration of 1mM and BCF concentration of 0.5mM, and (B) immunoblotting analysis shows the protein expression of sfGFP-N150BCF under different concentrations of BCF. CB-Coomassie brilliant blue staining.

FIG. 17 SDS-PAGE and mass spectrometry analysis of cpsfGFP-Y163BCF protein. (A) SDS-PAGE electrophoresis of purified cpsfGFP-Y163BCF protein; (B) ESI-MS mass spectrometry of cpsfGFP-Y163BCF protein;

FIG. 18 fluorescence response of negative control cpsfGFP-Y163BCF to OH in vitro. (A) The fluorescence response of 0.5 mu McpsfGFP-Y163HBCF probe and negative control cpsfGFP-Y163BCF to different concentrations of OH, the excitation wavelength is 470nm, the emission wavelength is 510nm, and the fluorescence response spectrum of 0.5 mu M negative control cpsfGFP-Y163BCF to different concentrations of OH, the excitation wavelength is 470nm. Incubation time was 60min and data were shown as mean ± standard deviation (n=3).

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Partial term definition

Unless defined otherwise hereinafter, all technical and scientific terms used in the detailed description of the invention are intended to be identical to what is commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.

As used herein, the terms "comprising," "including," "having," "containing," or "involving," are inclusive (inclusive) or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of" is considered to be a preferred embodiment of the term "comprising". If a certain group is defined below to contain at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.

The indefinite or definite article "a" or "an" when used in reference to a singular noun includes a plural of that noun.

The term "about" in the present invention means a range of accuracy that one skilled in the art can understand while still guaranteeing the technical effect of the features in question. The term generally means a deviation of + -10%, preferably + -5%, from the indicated value.

Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The following terms or definitions are provided solely to aid in the understanding of the invention. These definitions should not be construed to have a scope less than understood by those skilled in the art.

As used herein, the term ". OH-reactive" refers to a property of specifically reacting with. OH. Reaction is also a chemical term that refers to the process of interaction between two or more chemicals to produce a new chemical, i.e., chemical reaction (chemical reaction).

As used herein, the term "associate" refers to a component that acts in concert, such as an orthogonal tRNA and an orthogonal aminoacyl-tRNA synthetase. These components may also be referred to as "complementary" to each other.

As used herein, the term "homologous" refers to the concept of homology in genetics, which refers primarily to sequence homology, indicating that two or more protein or DNA sequences have the same ancestor. Homologous sequences are also likely to have similar functions. The two sequences are either homologous or different, and there is no concept of "homology". The homologous portions of the sequences are also known as conserved (conserved).

As used herein, the term "cognate tRNA" refers to cognate tRNA's that are homologous and co-act with a translation system of interest, and it is to be understood that virtually any amino acid (whether natural or unnatural) can be incorporated into an extended polypeptide, preferably by utilizing the cognate tRNA's of the invention to react with a selector codon during translation by the translation system.

As used herein, the term "pyrrolysinyl-tRNA synthetase" refers to an enzyme that catalyzes the activation of pyrrolysine and is covalently bound to the 3' end of a corresponding tRNA molecule. Pyrrolysine (Pyl) is found in methanogen methylamine methyltransferase, the 22 nd amino acid known to date to be involved in protein biosynthesis. Unlike the standard amino acids, pyrrolysine (Pyl) is formed by the sense coding of the stop codon UAG. Correspondingly, specific pyrrolysinyl-tRNA synthetases (PyleSs) and pyrrolysine tRNA (tRNA ^Pyl) are also contained in methanogens, which have specific structures that differ from classical tRNA's. Methanogens produce pyrrolysinyl-tRNA ^Pyl(Pyl-tRNA^Pyl via direct and indirect pathways, which may control UAG encoding to a stop codon or pyrrolysine through specific structures on mRNA and other mechanisms not yet found. Wild-type pyrrolysinyl-tRNA synthetases (PyleSs) are available, for example, from Methanopyrrococcus equi (Methanosarcinamazei), methanopyrrococcus barbites (Methanosarcinabarkeri), methanopyrrococcus acetate (Methanosarcina acetivorans), and the like, as methanogenic archaea.

The term "aminoacyl-tRNA synthetase" as used herein refers to a mutant that is introduced by various methods based on a wild-type pyrrolysiyl-tRNA synthetase (PyleS). The mutant pyrrolysinyl tRNA synthetase HBCFRS of the present invention is a "mutant pyrrolysinyl tRNA synthetase", and the mutant Pyles (aRS, bRS) of the present invention can efficiently introduce a protein even in the case of an unnatural amino acid HBCF that is inactive when the wild-type Pyles is used.

The term "unnatural amino acid" as used herein refers to any amino acid, modified amino acid and/or amino acid analog that is not among the 20 common natural amino acids. For example, the present invention may use the unnatural amino acid HBCF.

The term "affinity purification" as used herein refers to a method of purifying proteins by separating and purifying biological substances using the principle of specific binding between biological molecules.

The term "translation system" as used herein refers to the incorporation of amino acids into the components of an extended polypeptide chain (protein). Components of the translation system can include, for example, ribosomes, tRNA's, synthetases, mRNA, and the like. The O-tRNA's and/or O-RSs of the invention can be added to or part of an in vitro or in vivo translation system, e.g., in a non-eukaryotic cell, e.g., a bacterium (e.g., E.coli), or in a eukaryotic cell, e.g., a yeast, mammalian cell, plant cell, algal cell, fungal cell, insect cell, etc.

The term "PBS" as used herein refers to phosphate buffered saline, phosphate buffer saline, which is well known and widely used by those skilled in the art.

The invention is further described by the accompanying drawings and the following examples, which are provided to illustrate specific embodiments of the invention and are not to be construed as limiting the scope of the invention in any way.

Detailed Description

EXAMPLE 1 design and preparation of OH (hydroxyl radical) reactive unnatural amino acid HBCF

The synthetic route for HBCF is shown in figure 2. The present invention studied the synthesis of HBCF starting from commercially available N.alpha. -Boc-4-amino-L-phenylalanine (N.alpha. -Boc-4-amino-L-phenylalanine, boc-AF, compound 1) and methyl 3, 5-dihydroxybenzoate (3, 5-Dihydroxy-2-methylbenzoate, compound 3). To increase the yield, the carboxylic acid group of compound 1 is first protected with methyl ester to give compound 2. The compound 3 is protected by tert-butyl dimethyl chlorosilane to obtain a compound 4. The compound 4 is reduced by lithium aluminum hydride to obtain a compound 5, and then the compound 5 is reacted with triphosgene and then reacted with the compound 2 to generate a compound 6. And deprotecting the compound 6 by tetrabutylammonium fluoride, lithium hydroxide and trifluoroacetic acid in sequence to obtain the unnatural amino acid HBCF.

The specific preparation method of the HBCF comprises the following steps (see figure 2):

Compound 2 Is a synthesis of (a).

Compound 1 (1.85 g,6.59 mmol) was dissolved in methanol (40 mL) and cooled to 0deg.C, and thionyl chloride (785 mg,6.59 mmol) was slowly added dropwise. The reaction was slowly brought to room temperature and stirred for 2.5hr. After the reaction was completed, most of methanol was removed by vacuum concentration by vacuum pump, then dissolved in ethyl acetate and washed with saturated sodium bicarbonate and brine. Finally, the mixture is dried by anhydrous sodium sulfate solid and filtered, and the filtrate is decompressed and concentrated by a vacuum pump to obtain a yellow oily liquid compound 2-2(1.92g,96％).1HNMR(300MHz,CDCl3)δ6.89(d,J＝8.3Hz,2H),6.61(d,J＝8.4Hz,2H),4.95(d,J＝7.7Hz,1H),4.50(dd,J＝13.7,5.9Hz,1H),3.70(s,3H),2.99–2.95(m,2H),1.41(s,9H).13C NMR(75MHz,CDCl3)δ172.70,155.28,145.45,130.25,125.75,115.41,79.94,54.70,52.25,37.56,28.42.HRMS calcd for C15H22N2O4[M+H]+:295.1658;found:295.1650.

Compound 4Is a synthesis of (a).

Compound 3 (1 g,5.95 mmol) was dissolved in DCM (40 mL) and then triethylamine (1.44 g,14.27 mmol), 4-dimethylaminopyridine (803 mg,2.97 mmol) and tert-butyldimethylchlorosilane (1.97 g,13.08 mmol) were added sequentially and stirred at room temperature for 12hr. After the reaction, the mixture was quenched with saturated ammonium chloride solution and extracted with DCM (3X 18 mL), and the organic phase was washed, dried and purified to give a colorless liquid compound 2-4(2.17g,92％).¹HNMR(300MHz,CDCl3)δ7.12(d,J＝2.3Hz,2H),6.52(t,J＝2.3Hz,1H),3.87(s,3H),0.98(s,18H),0.20(s,12H).13C NMR(75MHz,CDCl3)δ166.89,156.64,131.95,116.93,114.69,52.24,25.76,18.31.HRMScalcd for C20H36O4Si2[M+H]+:397.2230;found:397.2224.

Compound 5Is a synthesis of (a).

Compound 4 (2.17 g,5.49 mmol) was dissolved in THF (50 mL), lithium aluminum hydride (313 mg,8.24 mmol) was added in portions and stirred overnight at room temperature. After the reaction was completed, water was added thereto at 0 ℃. Filtering, concentrating, extracting with 100mL ethyl acetate, washing, drying, and purifying to obtain colorless liquid compound 5(1.31g,65％).¹HNMR(300MHz,CDCl3)δ6.46(d,J＝2.1Hz,2H),6.26(s,1H),4.53(s,2H),2.26(s,1H),0.98(s,18H),0.19(s,12H).13C NMR(75MHz,CDCl3)δ156.74,143.26,111.86,111.21,65.03,25.78,18.29.HRMS calcd for C19H36O3Si2[M+H]+:369.2281;found:369.2275.

Compound 6Is a synthesis of (a).

Compound 5 (1 g,2.72 mmol) was dissolved in DCM (40 mL) and triphosgene (322 mg,1.09 mmol) was added and stirred overnight at room temperature. Concentrating under reduced pressure, and directly feeding into the next step. Compound 2-2 (382 mg,1.30 mmol) was dissolved in DCM (20 mL), and triethylamine (390 mg,3.89 mmol), 4-dimethylaminopyridine (16 mg,0.13 mmol) and the product (1.12 g) after the reaction of the above 5 with triphosgene were added in this order and reacted at room temperature for 3hr. After the reaction is finished, the mixture is quenched by saturated ammonium chloride solution and extracted by DCM (3X 10 mL), and the organic phase is washed, dried and purified to obtain a white solid compound 2-6(714mg,80％).¹H NMR(300MHz,CDCl3)δ7.31(d,J＝8.0Hz,2H),7.05(d,J＝8.2Hz,2H),6.74(s,1H),6.48(s,2H),6.29(s,1H),5.05(s,2H),4.98(d,J＝7.7Hz,1H),4.54(d,J＝7.2Hz,1H),3.70(s,3H),3.10–2.95(m,2H),1.41(s,9H),0.97(s,18H),0.19(s,12H).13C NMR(75MHz,CDCl3)δ172.44,156.81,155.22,153.36,138.02,136.92,131.14,130.04,118.95,113.22,111.91,80.09,66.85,54.55,52.35,37.76,29.83,28.42,25.79,18.32.HRMScalcd forC35H56N2O8Si2[M+Na]+:711.3473;found:711.3469.

Compound 7Is a synthesis of (a).

Compound 6 (270 mg,0.39 mmol) was dissolved in THF (10 mL), tetrabutylammonium fluoride (1.18 mL,1.18 mmol) was added and reacted at room temperature for 2hr. After the reaction was completed, most of THF was removed by concentration under reduced pressure, and then dissolved in ethyl acetate (50 mL) and washed with saturated sodium bicarbonate solution, and a pale yellow solid compound was obtained by drying and purification 7(140mg,78％).¹HNMR(500MHz,d6-DMSO)δ9.67(s,1H),9.28(s,2H),7.37(d,J＝8.2Hz,2H),7.24(d,J＝8.0Hz,1H),7.13(d,J＝8.5Hz,2H),6.24(d,J＝2.2Hz,2H),6.15(t,J＝2.2Hz,1H),4.95(s,2H),4.14–4.09(m,1H),3.60(s,3H),2.93–2.76(m,2H),1.32(s,9H).13C NMR(126MHz,d6-DMSO)δ173.12,158.95,155.90,153.91,139.17,138.05,131.96,129.88,118.57,106.18,102.49,78.82,66.05,55.84,52.24,36.36,28.63.HRMS calcd for C23H28N2O8[M+Na]+:483.1743;found:483.1739.

Synthesis of Compound Boc-HBCF:

Compound 7 (110 mg,0.24 mmol) was dissolved in a mixed solution of 1mL of methanol and 1mL of tetrahydrofuran, and then a solution of lithium hydroxide (1.2 mg,4.78 mmol) in water (1 mL) was added thereto, and the reaction was carried out at room temperature for 40min. After the reaction was completed, 1M hydrochloric acid was added dropwise to a pH of about 2. Extraction with ethyl acetate (3X 10 mL) and washing, drying and purification of the organic phase gave the compound as a pale yellow solid Boc-HBCF(93mg,87％).¹HNMR(300MHz,d6-DMSO)δ9.68(s,1H),7.36(d,J＝8.3Hz,2H),7.14(d,J＝8.4Hz,2H),7.01(d,J＝8.2Hz,1H),6.23(d,J＝2.0Hz,2H),6.14(s,1H),4.95(s,2H),4.06–3.98(m,1H),2.97–2.71(m,2H),1.32(s,9H).13C NMR(75MHz,d6-DMSO)δ174.17,158.97,155.94,153.92,139.19,137.93,132.52,129.93,118.51,106.16,102.47,78.55,66.03,53.61,36.36,28.69.HRMS calcd for C22H26N2O8[M-H]-:445.1611;found:445.1618.

Synthesis of compound HBCF:

Compound Boc-HBCF (90 mg,0.20 mmol) was dissolved in DCM (1 mL) and trifluoroacetic acid (0.7 mL) was added and reacted at room temperature for 3hr. After the reaction was completed, excess trifluoroacetic acid was removed by concentration under reduced pressure, and then dissolved in water and washed three times with diethyl ether (5 mL). Freezing the water phase into solid with liquid nitrogen, and vacuum freeze drying to obtain yellowish solid compound HBCF(59mg,84％).¹H NMR(300MHz,CD3OD)δ7.46(d,J＝8.4Hz,2H),7.22(d,J＝8.5Hz,2H),6.33(d,J＝2.0Hz,2H),6.22–6.21(m,1H),5.02(s,2H),4.21(dd,J＝7.5,5.4Hz,1H),3.30–3.04(m,2H).13C NMR(75MHz,CD3OD)δ171.27,159.74,155.85,140.25,139.95,130.95,129.86,120.47,107.09,103.14,67.46,55.14,36.69.HRMS calcd for C17H18N2O6[M-H]-:345.1087;found:345.1093.

Example 2 LC-MS analysis experiment of HBCF reacting with OH

After completion of the synthesis of HBCF, the present invention intends to verify its reaction with OH by liquid chromatography-mass spectrometry (LC-MS). According to the reaction mechanism (FIG. 1), OH reacts with the DHBC group of HBCF, releasing AF. To obtain the appropriate retention time in the column, detection was performed with a tert-butoxycarbonyl (Boc) protected HBCF derivative, i.e., boc-HBCF, which was expected to react with OH to produce Boc-AF (FIG. 3).

Boc-HBCF was reacted with 100 equivalents of OH at 37℃for 1hr, followed by LC-MS analysis. As shown in FIG. 4A, the retention time of Boc-HBCF in HPLC was 5.34min, while the retention time of Boc-AF in HPLC was 3.69min. In the reaction solution of Boc-HBCF and OH, only a chromatographic peak having a retention time of 3.69min was detected in the HPLC analysis, which was consistent with the retention time of Boc-AF. Mass spectrometry showed that the molecular weight of the product at 3.69min after the reaction was also consistent with Boc-AF (fig. 4D, E).

This result demonstrates that the product after the reaction has the same retention time and molecular weight as Boc-AF, i.e., boc-HBCF reacts with OH to do produce Boc-AF. From this, it was inferred that HBCF reacts with OH to form AF after site-directed insertion of protein.

EXAMPLE 3 site-directed insertion of HBCF into E.coli

1) Screening for synthase mutants recognizing HBCF

The present invention models the HBCF computational simulation into the pyrrolysine binding pocket of pyrrolysine HBCF synthetases in order to obtain active site mutants of pyrrolysiyl tRNA synthetases capable of inserting HBCF, and notes that the side chain of HBCF may spatially collide with many residues around the pocket (e.g., Y306, L309, C348, M350, I405 and I413V). Thus, by rational design of mutations of these residues to less sterically hindered amino acids, a series of active site mutants based on wild-type pyrrolysinyl tRNA synthetases were constructed. The results of the screening experiments of the synthetase mutants are shown in FIG. 5, two (aRS, bRS) of 26 synthetase mutants can effectively identify the HBCF and insert the HBCF into the Y39 site of the EGFP, so that a stronger EGFP protein fluorescent signal is displayed, and a control group without the HBCF has no obvious EGFP protein fluorescent signal. This also demonstrates that the system has good orthogonality with the natural amino acid synthase system in E.coli.

To screen for synthetase mutants, the pLX-EGFP-Y39TAG plasmid was first co-transformed with the constructed pBX-MmPylRS active site mutant plasmid, respectively, into BL21 (DE 3) E.coli competent cells, the transformed monoclonal colonies were picked, and shake-cultured overnight at 37℃on LB medium containing chloramphenicol (34. Mu.g/mL) and kanamycin (40. Mu.g/mL). The overnight cultured strain was diluted at a ratio of 1:100 and inoculated into fresh LB medium containing antibiotics. When incubated at 37℃to an OD600 of 0.6, 1mM UAA (HBCF or BCF) was added. After 1hr of incubation at 37 ℃, 1mM IPTG was added to induce protein expression. After culturing at 37℃for 9hr, bacteria were collected, and finally the fluorescence intensity of the bacterial liquid was measured with a Biotek Synergy H1 microplate reader and compared with a blank group to which UAA was not added.

It is appreciated that two mutant pyrrolysinyl tRNA synthetases recognizing HBCF are found (aRS, bRS) in FIG. 5, and can be efficiently introduced into EGFP-Y39TAG to recognize HBCF.

2) ARS, bRS insertion efficiency experiment on HBCF

Proteins were transferred from SDS-PAGE gels onto nitrocellulose membranes using a full-format semi-dry transfer instrument (25V, 30 min) and blocked in PBST-milk (5% skimmed milk, 0.05% Tween-20) for 1hr at room temperature. The nitrocellulose membrane was then incubated with the corresponding antibody solution overnight at 4 ℃, washed 3 times with PBST (containing 0.05% tween-20), developed with CLARITY WESTERN ECL luminophores and finally imaged with a ChemiDoc all-purpose gel imager. The intensities of the bands in the fluorescent gel and Western blot were subsequently quantified by Image Lab software (Bio-Rad). .

In general, sfGFP is expressed in a higher amount than EGFP, and insertion of UAA into the sfGFP-N150 site does not affect fluorescence intensity, so the present study selected sfGFP for subsequent characterization experiments. As shown in FIG. 6A, the immunoblot analysis shows that under the experimental conditions, the sfGFP full-length protein containing HBCF, i.e., sfGFP-N150HBCF, can be expressed more highly under the recognition of the synthetase mutant aRS (MmPylRS-Y306A/N346A/C348A/Y384F/W417T, hereinafter referred to as HBCF-RS), so that the subsequent experiments all use HBCF-RS as the synthetase of HBCF. In addition, the present inventors studied the insertion efficiency of HBCF with the well-known insertion of the excellent substrate of N.alpha. -Boc-L-lysine (N.alpha. -Boc-L-lysine, bocK) of wild-type MmPylRS as a positive control. As shown in FIG. 6C, immunoblot analysis showed that sfGFP-N150HBCF was able to exhibit an insertion efficiency comparable to BocK under the experimental conditions. Meanwhile, concentration gradient experiments of HBCF show that the expression level of sfGFP-N150HBCF protein is obviously improved along with the increase of the concentration of HBCF (figure 6D), and the fluorescence intensity of protein is also obviously increased (figure 6E). .

3) Purified sfGFP-N150HBCF protein

The pLX-cpsfGFP-Y163TAG-Twin-Strep II plasmid was co-transformed with pBX-HBCF-RS or pBX-BCF-RS plasmid into E.coli BL21 (DE 3), the transformed monoclonal colonies were picked and shaken overnight under antibiotic-containing LB culture conditions based on 37 ℃. The overnight cultured strain was then diluted 1:100 and inoculated into fresh LB medium containing antibiotics. When incubated at 37℃to an OD600 of 0.6, 2mM HBCF or 0.5mM BCF was added. After 1hr of incubation at 37 ℃,1mM IPTG was added to induce protein expression. After culturing at 37℃for 9hr, the bacteria were collected by centrifugation. In the protein expression analysis experiments, the collected bacteria were lysed with cell lysates (50 mM TEA, 150mM NaCl, 4% SDS, pH 7.4) at 95℃and then incubated with 0.2. Mu.L nuclease at room temperature for 10min, and centrifuged (16,000Xg, 5 min) to remove cell debris. Protein concentration was determined using BCA method. After addition of 4 Xloading buffer, incubation is carried out for 5min at 95℃to separate the cell lysates on SDS-PAGE gels, and finally the experimental results are analyzed by Coomassie blue staining and immunoblotting. In preparing the purified protein, bacteria were sonicated in Buffer W (100 mM Tris-HCl,1mM EDTA,150mM NaCl,pH 8.0) containing lysozyme, DNaseI, protease inhibitors and phenylmethylsulfonyl fluoride (Phenylmethylsulfonyl fluoride, PMSF). Then high-speed centrifuging, collecting supernatant andThe XT resin was incubated for 2hr, then washed with BufferW and eluted with Buffer BXT (Buffer W with 50mM biotin), and finally Buffer W was replaced with PBS Buffer by dialysis. The whole process of preparing purified protein is carried out on ice or under the condition of 4 ℃ and stored at-80 ℃. The antibiotic formulation was 34. Mu.g/mL chloramphenicol and 40. Mu.g/mL kanamycin.

As shown in FIG. 7, SDS-PAGE shows that the sfGFP-N150HBCF protein prepared and purified from E.coli had a band of about 31kDa, which corresponds to its theoretical molecular weight of 31.2kDa (FIG. 7A). Electrospray ionization mass spectrometry (ESI-MS) analysis showed a protein molecular weight of 31212.03Da, which was substantially consistent with theoretical 31212.76Da (FIG. 7B). These results demonstrate that HBCF can be inserted into the target protein in E.coli by the HBCF-RS/tRNAPCyUlA orthogonal pair with high efficiency and high fidelity.

The result shows that the unnatural amino acid HBCF can be inserted into target protein in escherichia coli at fixed points through a genetic code expansion technology.

Example 4 preparation of OH fluorescent Probe cpsfGFP-Y163HBCF and in vitro OH detection

1) Construction of OH-fluorescent Probe cpsfGFP-Y163PHBCF preparation

After the fixed-point insertion of the HBCF is successfully realized, the invention researches the key Tyr locus of the fluorescent protein chromophore introduced by the HBCF, after the HBCF is inserted into the locus, the fluorescent intensity of the fluorescent protein is expected to be influenced by a side chain with larger volume of the HBCF, and when the HBCF reacts with OH to remove the DHBC group of the side chain, the fluorescence can be recovered. First, the expression levels of several commonly used green fluorescent proteins EGFP, sfGFP, cpsfGFP inserted into UAA and red fluorescent protein mCherry in E.coli were compared, and it was desired to find the fluorescent protein with the highest expression level in order to perform the subsequent in vitro reaction. Wherein, EGFP, sfGFP, cpsfGFP green fluorescent proteins are all engineered from GFP, EGFP and sfGFP have sequences with extremely high similarity (> 90%) with GFP, cpsfGFP is a cyclic GFP formed by linking the N-terminal and C-terminal ends of sfGFP and GFP, the similarity with GFP sequences is less than 60%, tyr in chromophore is still present although its primary structure amino acid sequence varies greatly, and has been reported for fixed point insertion of UAA. mCherry is a red fluorescent protein from mushroom corals, and although less than 25% similar to GFP sequence, still has the same properties as GFP, i.e. fluorescent signal is affected by key sites of chromophore.

Immunoblot analysis revealed that the introduction of HBCF at the chromophore tyrosine site of several fluorescent proteins expressed the full-length protein, and that cpsfGFP-Y163HBCF expressed the highest amount (FIG. 8A). Further HBCF concentration gradient experiments show that the cpsfGFP-Y163HBCF protein can achieve better expression effect when the concentration of the HBCF is 2mM (figure 8B), so that the concentration of 2mM is selected to express cpsfGFP-Y163HBCF protein in escherichia coli, and the HBCF protein is used as a potential OH fluorescent probe for subsequent in vitro detection.

The prepared purified protein was desalted by a centrifugal filter and redissolved in ultrapure water. The proteins were then mass analyzed using a Nano-ESI ionization source and Q Exactive LC-MS/MS system (Thermo Scientific) and the resulting data were deconvolved with BioPharma Finder software (Thermo Scientific). As shown in FIG. 9, SDS-PAGE results show that the band of cpsfGFP-Y163HBCF protein prepared from E.coli was about 30kDa and showed a good purity. ESI-MS analysis showed that the protein molecular weight was 30447.41Da, which was substantially consistent with theoretical 30446.81 Da. These results verify the integrity and stability of the probe for subsequent OH detection.

2) In vitro OH detection of OH fluorescent probe cpsfGFP-Y163HBCF

In a 96-well bottom-penetration blackboard, 0.5. Mu.M cpsfGFP-Y163HBCF or cpsfGFP-Y163BCF protein was incubated with different concentrations of OH for 60min at room temperature. The fluorescence spectrum of the protein of each well at 470nm excitation or the emitted fluorescence intensity at 510nm was measured by an enzyme-labeled instrument. In testing the selectivity of the probes, 0.5. Mu. McpsfGFP-Y163HBCF protein was incubated with 5. Mu.M.OH or 50. Mu.M other possible interferents (preparation methods see 2.4.4.1) at room temperature for 60min, then the same experimental procedure was performed and three biological replicates were performed. The experiment was performed in 20mMPBS buffer (pH 7.4).

The results show that, as shown in FIG. 10, a significant increase in fluorescence signal was observed at 510nm of the maximum emission peak with increasing OH concentration at 470nm excitation, and concentration dependence was exhibited, both for TCBQ/H2O2 system and for LH/H2O2 system. Since the experimental procedure for the preparation of TCBQ/H2O2 system is simpler, if not specified, the OH of the subsequent experiments were all generated by TCBQ/H2O2 system ([ H2O2]: TCBQ ] = 10:1, [. OH ] = [ TCBQ ]).

3) EcpsfGFP-Y163HBCF probe detects the sensitivity and kinetic response of OH.

For further validation, the present invention performed statistical analysis of fluorescence response results for EcpsfGFP-Y163HBCF probe at a concentration range of 0-10. Mu.M. OH. As shown in FIG. 11A, cpsfGFP-Y163HBCF probe showed good linear correlation between fluorescence intensity at 510nm and OH concentration of 0-4. Mu.M (R2= 0.9906). Statistical analysis of the fluorescence response results for OH at a concentration range of 0-1. Mu.M revealed that the in vitro limit of detection of OH by this probe was about 1. Mu.M (FIG. 11B). Furthermore, kinetic analysis showed that the fluorescence-on response of cpsfGFP-Y163HBCF to OH was substantially saturated around 60min (FIG. 11C).

4) Experiments of cpsfGFP-Y163HBCF probe vs. OH response

Specifically, the probe response to various potential interferents (TBCQ, H2O 2) and intracellular common ROS (e.g., O2-, 1O2, NO, ONOO-, HOCl, etc.) was tested by incubating cpsfGFP-Y163HBCF (0.5. Mu.M) with 50. Mu.M interferents for 60min at room temperature, followed by fluorescence detection. As shown in FIG. 12, cpsfGFP-Y163HBCF probe showed strong fluorescence response only in the presence of OH, and was essentially non-responsive to other interferents, indicating good selectivity of cpsfGFP-Y163HBCF probe to OH.

In addition, to verify the response mechanism of cpsfGFP-Y163HBCF probe to OH, the present study analyzed the protein molecular weight of cpsfGFP-Y163HBCF after OH treatment using ESI-MS. As shown in FIG. 13, the molecular weight of the protein after reaction with OH was 30300.40Da, which was substantially the same as the theoretical molecular weight of the protein after removal of DHBC groups, 30300.78 Da. This result indicated that cpsfGFP-Y163HBCF did react with OH to remove the DHBC groups, yielding cpsfGFP-Y163AF.

Example 5 in vitro reaction of negative control cpsfGFP-Y163BCF with OH

To further verify the mechanism by which the cpsfGFP-Y163HBCF probe detects OH, the present study synthesized the unnatural amino acid 4- (benzyloxycarbonylamino) -L-phenylalanine (4- (Benzyloxycarbonylamino) -L-phenalanine, BCF) that was structurally similar to HBCF but did not contain an OH reactive group, and it was desirable to prepare the cpsfGFP-Y163BCF protein in the same manner as a negative control for the cpsfGFP-Y163HBCF probe.

1) The synthetic route for BCF is shown in fig. 14. The compound 2 obtained in example 1 was reacted with commercially available benzyl chloroformate (Benzyl chlorofomate, cbzCl) to give compound 8. And the compound 8 is subjected to deprotection by lithium hydroxide and trifluoroacetic acid in sequence to obtain the unnatural amino acid BCF.

1.1 Compound 8)Is synthesized by the following steps:

Compound 2 (1.5 g,5.10 mmol) was dissolved in DCM (50 mL), triethylamine (1.03 g,10.20 mmol), 4-dimethylaminopyridine (61 mg,0.50 mmol) and benzyl chloroformate (870 mg,5.10 mmol) were added sequentially and reacted at room temperature for 3hr. After the reaction, the mixture was quenched with saturated ammonium chloride solution, extracted with DCM (3X 15 mL), and the organic phase was washed, dried and purified to give a white solid compound 2-8(1.1g,50％).¹HNMR(500MHz,CDCl3)δ7.37–7.30(m,7H),7.23(s,1H),7.02(d,J＝8.4Hz,2H),5.17(s,2H),5.07(d,J＝8.1Hz,1H),4.56–4.52(m,1H),3.66(s,3H),3.05–2.95(m,2H),1.41(s,9H).13CNMR(126MHz,CDCl3)δ

172.39,155.19,153.51,137.05,136.21,130.94,129.82,128.57,128.27,128.23,118.90,79.97,66.90,54.53,52.16,37.61,28.30.HRMS calcd for C23H28N2O6[M-H]-:427.1869;found:427.1875.

1.2 Compound 9)Is synthesized by the following steps:

Compound 8 (960 mg,2.24 mmol) was dissolved in a mixed solution of 2mL of methanol and 2mL of THF, and then a solution of lithium hydroxide (109 mg,4.48 mmol) in water (3 mL) was added and reacted at room temperature for 40min. After the reaction was completed, 1M hydrochloric acid was added dropwise to a pH of about 2. Extraction with ethyl acetate (3X 15 mL) and washing, drying and purifying the organic phase to give the white solid compound 2-9(848mg,91％).¹H NMR(500MHz,CDCl3)δ7.34–7.32(m,7H),7.17(s,1H),7.08(d,J＝8.3Hz,2H),5.17(s,2H),5.02(d,J＝7.6Hz,1H),4.60–4.59(m,1H),3.10–2.90(m,2H),1.43(s,9H).13C NMR(126MHz,CDCl3)δ176.07,155.51,153.89,137.10,136.07,131.07,130.19,128.72,128.46,128.42,119.12,80.41,67.27,54.42,37.25,28.44.HRMS calcd for C22H26N2O6[M-H]-:413.1713;found:413.1716.

1.3 Synthesis of compound BCF:

Compound 9 (840 mg,2.03 mmol) was dissolved in DCM (2 mL) and trifluoroacetic acid (2 mL) was added and reacted at room temperature for 3hr. After the reaction was completed, excess trifluoroacetic acid was removed by concentration under reduced pressure, and then dissolved in water and washed three times with diethyl ether (20 mL). Freezing the water phase into solid with liquid nitrogen, and vacuum freeze drying to obtain pale white solid compound BCF(503mg,79％).¹H NMR(500MHz,d6-DMSO)δ9.70(s,1H),7.41–7.36(m,7H),7.15(d,J＝8.3Hz,2H),5.14(s,2H),3.07(dd,J＝14.3,4.2Hz,1H),2.84–2.71(m,1H).13C NMR(126MHz,d6-DMSO)δ170.60,153.92,138.77,137.15,130.34,129.13,128.93,128.52,119.34,66.23,53.80,35.74.HRMS calcd forC17H18N2O4[M-H]-:313.1188;found:313.1195.

2) Screening of synthetic enzyme mutants recognizing BCF

The non-natural amino acid BCF is introduced into EGFP-Y39TAG by using a screening strategy using EGFP as a model protein, and the insertion efficiency of the synthetase mutant is screened in escherichia coli. As a result, as shown in FIG. 15, cRS (MmPylRS-Y306A/L309A/N346A/C348A/Y384F, named BCF-RS) was obtained by screening, and BCF was inserted into EGFP-Y39TAG to significantly increase the fluorescence signal of EGFP.

3) Site-directed insertion of BCF into E.coli

To examine the efficiency of BCF-RS recognition and insertion of BCF, BCF-RS was co-transformed with sfGFP-N150TAG plasmid into BL21 (DE 3) competent cells of E.coli. Immunoblot analysis showed that under this experimental condition, the sfGFP full-length protein containing BCF, sfGFP-N150BCF, was able to be expressed normally and exhibited an insertion efficiency comparable to BocK (fig. 16A). Further concentration gradient experiments showed that saturated expression levels were already achieved at a BCF concentration of 0.25mM (FIG. 16B). A concentration of 0.25mM was selected for expression and purification of cpsfGFP-Y163BCF in E.coli and used as a negative control for the cpsfGFP-Y163HBCF fluorescent probe.

CpsfGFP-Y163BCF protein was purified and further confirmed by protein mass spectrometry. As shown in FIG. 17, SDS-PAGE shows that the band of cpsfGFP-Y163BCF protein prepared from E.coli was about 30kDa, which corresponds to a molecular weight of 30.4 kDa. ESI-MS analysis showed that the protein molecular weight was 30414.23Da, which was substantially consistent with theoretical 30414.82 Da. This verifies the integrity and stability of cpsfGFP-Y163BCF protein.

The fluorescence signal was detected by incubating the cpsfGFP-Y163HBCF probe at 0.5. Mu.M and the negative control cpsfGFP-Y163BCF protein with different concentrations of OH at room temperature for 60min, respectively. As shown in FIG. 18A, the fluorescence intensity of probe cpsfGFP-Y163HBCF increased with increasing OH concentration, whereas the negative control cpsfGFP-Y163BCF did not have a significant fluorescence enhancement. In addition, no fluorescence emission of the negative control probe was detected in the 490nm-600nm spectral range (FIG. 18B). This further illustrates that probe cpsfGFP-Y163HBCF allows detection of OH by reaction of HBCF with OH.

Taken together, the results indicated that the genetically encoded OH probe cpsfGFP-Y163HBCF was able to selectively detect OH in vitro.

As described above, the present invention successfully developed a fluorescent probe which is genetically encoded based on cpsfGFP-Y163HBCF prepared from the same and can be used for detecting and imaging OH in a solution.

The above description of the embodiments of the present invention is not intended to limit the present invention, and those skilled in the art can make various changes or modifications according to the present invention without departing from the spirit of the present invention, and shall fall within the scope of the appended claims.

Claims (9)

1. An unnatural amino acid having the structural formula:

characterized by comprising a 4-amino-L-phenylalanine structure.

2. The unnatural amino acid of claim 1, wherein the unnatural amino acid is a genetically encoded hydroxyl radical reactive unnatural amino acid.

3. A hydroxyl radical reactive unnatural amino acid according to claim 1 or 2, wherein the method of preparing the unnatural amino acid comprises the steps of:

Step 1) protecting the carboxylic acid group of compound 1N α -t-butoxycarbonyl-4-amino-L-phenylalanine with methyl ester to obtain compound 2;

Step 2), protecting hydroxyl of 3, 5-dihydroxybenzoic acid methyl ester of the compound by tert-butyldimethyl chlorosilane to obtain a compound 4;

step 3) reducing the compound 4 by lithium aluminum hydride to obtain a compound 5;

Step 4) reacting the compound 5 with triphosgene and then reacting with the compound 2 to generate a compound 6;

And 5) sequentially deprotecting tetrabutylammonium fluoride, lithium hydroxide and trifluoroacetic acid to obtain the hydroxyl radical reactive unnatural amino acid.

4. A composition comprising the unnatural amino acid of claim 1.

5. An expression system, translation system comprising the unnatural amino acid of claim 1.

6. Use of a reactive unnatural amino acid according to claim 1 or 2 for the construction of a hydroxyl radical reactive fluorescent probe.

7. The hydroxyl radical fluorescent probe according to claim 6 is a genetically encoded fluorescent protein probe obtained by introducing the unnatural amino acid according to any one of claims 1 to 2 into a specific site of a biomacromolecule, wherein the probe is cpsfGFP-Y163 HBCF.

8. A composition comprising the hydroxyl radical reactive fluorescent probe of claim 6 or 7.

9. An expression system or translation system comprising the hydroxyl radical reactive fluorescent probe of claim 6 or 7.