CN111747869A

CN111747869A - Genetically encoded formaldehyde reactive unnatural amino acid, preparation method and application thereof

Info

Publication number: CN111747869A
Application number: CN202010538098.1A
Authority: CN
Inventors: 彭涛; 张雨晴; 杜一萌
Original assignee: Peking University Shenzhen Graduate School
Current assignee: Peking University Shenzhen Graduate School
Priority date: 2020-06-12
Filing date: 2020-06-12
Publication date: 2020-10-09
Anticipated expiration: 2040-06-12
Also published as: WO2021248770A1; CN111747869B

Abstract

The present invention relates to a genetically encoded formaldehyde-reactive unnatural amino acid. Specifically, the invention discloses a genetically encoded formaldehyde-reactive unnatural amino acid PrAK, a preparation method and application thereof in detection and imaging of formaldehyde. The formaldehyde reactive unnatural amino acid PrAK provided by the invention can be specifically introduced into green fluorescent protein (EGFP), firefly luciferase (fLuc) and other biological macromolecules at sites in the protein translation process to construct a reactive biological macromolecule formaldehyde fluorescent probe and a bioluminescent probe, and can effectively detect or image formaldehyde in a biological sample.

Description

Genetically encoded formaldehyde reactive unnatural amino acid, 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 formaldehyde in a biological sample.

Technical Field

Formaldehyde is the simplest Reactive Carbonyl Species (RCS), a well-known basic chemical feedstock widely used in industry and biomedicine, and is also a common environmental hazard and carcinogen. However, it is less well known that formaldehyde is produced in a series of biological processes in biological systems, including epigenetic regulation, one-carbon unit metabolic energy, alcohol detoxification, and the like. For example, lysine demethylase (LSD) or histone demethylase (JmjC domain-conjugatinghistadimethylases, JHDM) can produce formaldehyde by-products by catalyzing the demethylation of histones; semicarbazide-sensitive amine oxidase (SSAO) catalyzes the deamination of methylamine to form formaldehyde in one-carbon metabolism; in addition, methanol can be oxidized by alcohol dehydrogenase1 (ADH 1) to form formaldehyde in cells. In addition to various endogenous formaldehyde sources, there are also corresponding molecular mechanisms in organisms for the breakdown of formaldehyde, for example by oxidation to formic acid by the action of aldehyde dehydrogenase (ALDH 2) or alcohol dehydrogenase 3 (ALDH 3, ADH3, also known as ADH 5). Therefore, under physiological conditions, the concentration of formaldehyde in the organism is maintained at a steady level, such as a concentration of approximately 0.1mM in blood or urine. And approximately 0.2-0.4mM in the brain and cells. Physiological levels of endogenous formaldehyde are an important source of carbon that can be used to make important cellular building blocks and mediate normal physiological processes as signaling molecules. However, abnormal increases in endogenous formaldehyde concentrations in the body have been reported to be associated with the development of a range of diseases, including cancer, Alzheimer's disease, stroke and diabetes. In fact, the concentration level of formaldehyde in urine of moderate and severe dementia patients is more than three times that of the healthy control group, and reaches 0.3-0.4 mM; it is estimated that formaldehyde concentrations in cancer tissues can be as high as 0.8 mM. However, the precise role of formaldehyde in physiological and pathological processes is still poorly understood, which has prompted ongoing efforts to develop new tools for detecting formaldehyde in biological samples.

Although conventional detection methods including colorimetric, chromatographic and radiological assays can be used to measure formaldehyde levels in plasma and homogenized tissues, these methods require complex sample handling procedures that disrupt sample integrity, limiting the use of non-invasive and in situ formaldehyde detection in living biological samples. Optical probes, especially fluorescent probes and bioluminescent probes, have the advantages of non-invasiveness, real-time performance, space-time imaging capability and the like, and have become a powerful alternative for formaldehyde detection. Therefore, the development of formaldehyde optical probes (including formaldehyde fluorescent probes and bioluminescent probes) is crucial to detecting and imaging the formaldehyde content in biological samples and revealing the role of formaldehyde in physiological and pathological conditions. Over the past few years, a series of small molecule fluorescent and bioluminescent probes for formaldehyde have been developed. However, genetic coding formaldehyde fluorescent probes and bioluminescent probes based on proteins are rarely reported at present. Compared with small molecule optical probes, the genetic coding formaldehyde fluorescent probe and the bioluminescence probe based on the protein can be introduced into living cells or organisms in a DNA form, and have unique advantages, such as excellent cell retention, cell specificity, subcellular organelle specificity targeting, stable and durable markers and the like. In view of the above, the present invention discloses a genetically encoded formaldehyde-reactive unnatural amino acid PrAK, which is site-specifically introduced into proteins, such as Enhanced Green Fluorescent Protein (EGFP) and firefly luciferase (ffluc), for constructing protein-based genetically encoded formaldehyde fluorescent probes and bioluminescent probes, respectively, for selective detection and imaging of formaldehyde in biological samples.

The key to the construction of genetically encoded formaldehyde fluorescent and bioluminescent probes using the formaldehyde-reactive unnatural amino acid PrAK is the site-specific insertion into the protein. Proteins are generally composed of 20 natural amino acids. As a template for protein synthesis, the sequence of nucleotides (A, U, C, G) on mRNA determines the amino acid sequence of the protein. Three nucleotides on mRNA form a triplet code (triplet code) that determines one amino acid. The triplet code for 4 nucleotides A, U, C, G is 64, 61 of which are used to encode 20 standard amino acids, and 3 are: UAA, UAG, UGA are typically stop codons, which direct the termination of protein translation. The art has developed a set of "orthogonal translation systems" that allow site-specific insertion of various unnatural amino acids into proteins in both prokaryotic and eukaryotic organisms. This system relies on an exogenous orthogonal protein translation component that recognizes an appropriate selector codon (e.g., the stop codon UAG, i.e., TAG on the corresponding DNA) during translation of the polypeptide in vivo, while inserting the desired unnatural amino acid into the prescribed position. This system utilizes an orthogonal tRNA (O-tRNA) that recognizes a selector codon (e.g., the stop codon UAG, i.e., TAG on the corresponding DNA), and a corresponding specific orthogonal aminoacyl-tRNA synthetase (O-RS) that charges the O-tRNA with an unnatural amino acid. These components do not cross-react (i.e., it must be orthogonal) with any endogenous tRNA, aminoacyl-tRNA synthetase (RS), natural amino acid, or codon encoding a natural amino acid in the host organism. This exogenous, orthogonal tRNA-RS pair can be used to genetically encode a large number of structurally diverse unnatural amino acids.

Those skilled in the art are familiar with how to utilize "orthogonal translation systems" suitable for preparing proteins containing unnatural amino acids, as well as general methods for generating orthogonal translation systems. See, for example, International publication No. WO2002/086075 entitled "Methods and compositions for generating Orthogonal tRNA-Aminoacyl-tRNA Synthetase Pairs" (Methods and compositions for the production of Orthogonal tRNA-Aminoacyl-tRNA synthetases); WO2002/085923 entitled "In Vivo Incorporation of Unnatural Amino Acids" (In Vivo Incorporation of Unnatual Amino Acids); WO2004/094593 entitled "expanded Eukaryotic genetic code" (Expanding the Eukaryotic genetic code); WO2005/019415 entitled "orthogonal translation Components for In Vivo Incorporation of Unnatural amino acids" (ortho translation Components for the In Vivo Incorporation of Unnatual amino acids). Additional discussion of orthogonal translation systems incorporating unnatural amino acids and methods for their production and use can also be found in the following papers: wang and Schultz, "extended genetic Code" (Expanding the genetic Code), chem. Commun. (Camb.)1:1-11 (2002); wang and Schultz, "extended the Genetic Code," Angewandte Chemie int. Ed.44(1):34-66 (2005); xie and Schultz, "extended Genetic Code" (Anexpansion Genetic Code), Methods 36(3): 227-; xie and Schultz, "Add Amino Acids to the Genetic library" (Adding Amino Acids to the Genetic repleroire), curr. opinion in chemical Biology 9(6):548-554 (2005); wang et al, "expanded genetic Code" (Expanding the genetic Code), Annu.Rev.Biophys.Biomol.Structure.35: 225-249 (2006); xie and Schultz, "chemical tool kit for Proteins-extended Genetic Code" (analytical Toolkit for Proteins-and expanded Genetic Code), nat. Rev. mol. cell biol.7(10):775-782 (2006).

Disclosure of Invention

The invention aims to provide an unnatural amino acid (a lysine analogue) capable of reacting with formaldehyde and a method for constructing a protein-based genetic code formaldehyde fluorescent probe or bioluminescent probe by using the unnatural amino acid. The present invention envisages the synthesis of an unnatural amino acid (unnatural amino acid) with formaldehyde reactivity specifically inserted into biomacromolecule fluorescent or bioluminescent proteins to control their fluorescence and bioluminescence, respectively (fig. 1A, 1B). The unnatural amino acid-modified EGFP and ffluc mutants are free of fluorescence and bioluminescence, respectively, when formaldehyde is not present; in the presence of formaldehyde, the unnatural amino acid reacts with formaldehyde to revert to a critical lysine residue, resulting in restoration of its fluorescence and bioluminescence (fig. 1A, 1B), thereby enabling formaldehyde detection.

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

the invention provides an unnatural amino acid lysine analogue capable of reacting with formaldehyde, which has the following structural general formula:

wherein R is alkyl; preferably, R is methyl, ethyl, propyl or benzyl.

In particular, such an unnatural amino acid is a lysine analog comprising L-lysine, a homoallylamine group that reacts with formaldehyde, and a nitrogen substituted alkyl group. The L-lysine is subjected to addition reaction with formaldehyde and dehydration to generate imine positive ions, then 2-azacophographic rearrangement reaction is carried out to generate a new imine positive ion, and then L-lysine is finally obtained through further hydrolysis and beta-elimination.

The invention provides an unnatural amino acid lysine analogue PrAK capable of reacting with formaldehyde, which is characterized in that the structural formula of the unnatural amino acid PrAK is as follows:

specifically, the unnatural amino acid PrAK is a lysine analog comprising L-lysine, a homoallylamine group reactive with formaldehyde, and a nitrogen substituted propyl group. PrAK and formaldehyde are subjected to addition reaction and dehydration to generate imine positive ions, then 2-azacopropol rearrangement reaction is carried out to generate a new imine positive ion, and L-lysine is finally obtained through further hydrolysis and beta-elimination (shown in figure 1C).

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

step 1), carrying out addition reaction on 3-hydroxypropionaldehyde protected by hydroxyl, propylamine and allyl boronic acid pinacol ester to prepare homoallylamine;

step 2), carrying out amino protection on the homoallylamine, and removing a hydroxyl protecting group to generate an alcohol compound;

step 3), activating alcoholic hydroxyl groups of alcohol compounds, and connecting the alcohol compounds with amino-protected L-lysine to generate carbamate compounds;

and 4) removing the amino protecting group of the carbamate compound to generate the unnatural amino acid PrAK.

In some embodiments, the hydroxyl protecting group of step 1) is p-methoxybenzyl;

in some embodiments, the amine protecting group of step 2) is a tert-butyloxycarbonyl group (Boc);

in some embodiments, the activating of step 3) is activating alcoholic hydroxyl group with p-nitrophenyl chloroformate and the amine protecting group is t-butyloxycarbonyl (Boc) or fluorenylmethyloxycarbonyl (Fmoc).

Specifically, the addition reaction of p-methoxybenzyl protected 3-hydroxypropanal with propylamine and allylboronic acid pinacol ester produces homoallylamine, after which the amine group is protected with t-butyloxycarbonyl (Boc) and the p-methoxybenzyl protecting group is removed to produce alcohol. Next, the alcoholic hydroxyl group was activated with p-nitrophenyl chloroformate and reacted with Boc-protected L-lysine to generate a carbamate compound. Finally, the Boc protecting group is removed under acidic conditions to yield the unnatural amino acid PrAK, as shown in fig. 2. Fmoc-protected PrAK (Fmoc-PrAK) can be prepared by the same synthetic route as shown in FIG. 3, i.e. reacting p-nitrophenyl chloroformate activated alcohol with Fmoc-protected L-lysine to generate carbamate compound, and removing Boc protecting group to obtain Fmoc-PrAK.

Similarly, the unnatural amino acid lysine analogs where R is methyl, ethyl, or benzyl can also be prepared by similar synthetic methods.

The invention also provides a compound connected with the unnatural amino acid lysine analogue PrAK.

The invention also provides a composition comprising the above unnatural amino acid lysine analog PrAK.

The invention also provides an expression system, a translation system or a cell comprising the unnatural amino acid lysine analogue PrAK.

The invention also provides a biological macromolecule probe, which is characterized in that the biological macromolecule probe is obtained by introducing the unnatural amino acid lysine analogue PrAK into a specific site of a biological macromolecule.

In some embodiments, the biomacromolecule probe is a genetically encoded formaldehyde fluorescent probe and a bioluminescent probe, and is characterized by comprising biomacromolecules such as fluorescent protein and luciferase, wherein the non-natural amino acid lysine analogue PrAK is introduced into a specific site; preferably, the biomacromolecules such as the fluorescent protein and the luciferase are Enhanced Green Fluorescent Protein (EGFP) and firefly luciferase (fLuc);

in some embodiments, one of the critical lysine sites of the Enhanced Green Fluorescent Protein (EGFP) or firefly luciferase (ffluc) is specifically introduced with the unnatural amino acid PrAK; preferably, the key lysine site of the Enhanced Green Fluorescent Protein (EGFP) is K85; the key lysine site of the firefly luciferase (fLuc) is K529; more preferably, the genetically encoded formaldehyde fluorescent or bioluminescent probe is EGFP-K85PrAK and fLuc-K529 PrAK.

The invention also provides a composition containing the biological macromolecular probe.

The invention also provides an expression system, a translation system or a cell containing the biological macromolecular probe.

The invention also provides an application of the biological macromolecule or the genetically coded formaldehyde fluorescent probe or the bioluminescent probe in sample imaging or formaldehyde detection;

in some embodiments, the sample is a biological sample; preferably, the biological sample is a live experimental animal, such as a mouse, zebrafish, nematode, and drosophila; or the biological sample is a mammalian cell, such as a human HEK293T cell;

in some embodiments, the sample is an in vitro or extracellular sample.

The invention also provides a method for preparing a biomacromolecule probe by using the unnatural amino acid lysine analogue PrAK, which is characterized by comprising the following steps: and (3) introducing the unnatural amino acid lysine analogue PrAK to the biomacromolecule at a fixed point to prepare a biomacromolecule probe.

In some embodiments, the biological macromolecule is a genetically encoded formaldehyde fluorescent probe or bioluminescent probe characterized by: the unnatural amino acid lysine analogue PrAK is introduced into biomacromolecules such as fluorescent protein, luciferase and the like at fixed points to prepare a genetic code formaldehyde fluorescent probe or a bioluminescent probe.

Specifically, the method comprises co-expressing in a host cell, in the presence of an unnatural amino acid PrAK, a pyrrolysinyl-tRNA synthetase gene, a cognate tRNA, and a fluorescent protein or luciferase gene in which the site to which the unnatural amino acid PrAK is introduced is mutated to an amber stop codon TAG;

in some embodiments, further comprising a purification step, obtaining a genetically encoded formaldehyde fluorescent probe or bioluminescent probe by purification;

preferably, a fluorescent protein or luciferase protein incorporating PrAK at a specific site is prepared by affinity purification;

more preferably, by Ni, as is well known in the art²⁺Column or

The column performs affinity purification on the tag protein.

In some embodiments, the host cell is an e.coli cell, and the first and second expression vectors are transformed into e.coli simultaneously; the first expression vector contains a pyrrolysinyl-tRNA synthetase gene and a homologous correlation tRNA thereof, and the second expression vector contains a fluorescent protein or luciferase gene which is to be introduced with an unnatural amino acid PrAK and has a specific site mutated into an amber stop codon TAG. In a host cell, a fluorescent protein or luciferase gene with a specific site mutated into an amber stop codon TAG is translated, and the pyrrollysyl-tRNA synthetase gene and the homologous association tRNA thereof can specifically recognize the amber stop codon TAG, so that the site of the unnatural amino acid PrAK is specifically introduced into the fluorescent protein or luciferase protein. In a host cell, a newly expressed pyrrolysinyl-tRNA synthetase does not charge endogenous tRNA with a natural or unnatural amino acid at significant or, in some cases, detectable levels; on the other hand, aminoacyl-tRNA synthetases endogenous to the host cell do not charge newly expressed cognate tRNAs for pyrrolysinyl-tRNA synthetases with natural or unnatural amino acids.

In a particular embodiment, the host cell is, for example, E.coli BL21(DE 3).

In some embodiments, it is preferred that the site-directed introduction of the unnatural amino acid PrAK is performed using an orthogonal translation system consisting of a pyrrolysine-tRNA synthetase (PylRS) active mutant from Methanosarcina mazei, m, and its cognate tRNA. More preferably, the pyrrolysine-tRNA synthetase active mutant is a Y306A, L309A and Y384F mutant based on wild-type pyrrolysine-tRNA synthetase (MmPhylRS) of Methanosarcina mazei, which is named PrAKRS, and the amino acid sequence of the mutant is shown as SEQ ID NO. 1.

In some embodiments, the fluorescent protein is Enhanced Green Fluorescent Protein (EGFP), which has been specifically site-directed to the above-described unnatural amino acid lysine analog PrAK at lysine position 85 by the aforementioned "orthogonal translation system". In some embodiments, the luciferase is a firefly luciferase (ffluc) to which the above unnatural amino acid lysine analog PrAK has been specifically site-directed at the 529 th lysine site by the aforementioned "orthogonal translation system".

The invention also provides a method for imaging or detecting formaldehyde in a biological sample by using the unnatural amino acid lysine analogue PrAK, which is characterized by comprising the following steps:

the method comprises the following steps:

1) introducing the unnatural amino acid lysine analogue PrAK into biomacromolecules such as fluorescent protein, luciferase and the like at fixed points in a biological sample to express a genetically encoded formaldehyde fluorescent probe or a bioluminescent probe;

2) a genetically encoded formaldehyde fluorescent probe or bioluminescent probe is used to detect formaldehyde in a biological sample.

Specifically, the method comprises co-expressing in a biological sample a pyrrolysinyl-tRNA synthetase gene, a cognate tRNA, and a fluorescent protein or luciferase gene having a site to which an unnatural amino acid PrAK is introduced mutated to an amber stop codon TAG in the presence of the unnatural amino acid PrAK, thereby expressing in the biological sample the fluorescent protein or luciferase protein having PrAK introduced at the specific site, i.e., a genetically encoded formaldehyde fluorescent probe or bioluminescent probe.

In some embodiments, the biological sample is transformed into both the first and second expression vectors; the first expression vector contains a pyrrolysinyl-tRNA synthetase gene and a homologous correlation tRNA thereof, and the second expression vector contains a fluorescent protein or luciferase gene which is to be introduced with an unnatural amino acid PrAK and has a specific site mutated into an amber stop codon TAG. In a biological sample, a pyrrosinyl-tRNA synthetase gene and homologous association tRNA thereof can specifically recognize amber stop codon TAG in the translation process of a fluorescent protein or luciferase gene with a specific site mutated into amber stop codon TAG, so that an unnatural amino acid PrAK site is specifically introduced into the fluorescent protein or luciferase protein. In a biological sample, a newly expressed pyrrolysinyl-tRNA synthetase does not charge endogenous tRNA with a natural or unnatural amino acid at a significant level, or in some cases at a detectable level; on the other hand, endogenous aminoacyl-tRNA synthetases in a biological sample do not charge newly expressed cognate trnas for pyrrolysinyl-tRNA synthetases with natural or unnatural amino acids.

In some embodiments, the biological sample is a live experimental animal, such as a mouse, zebrafish, nematode, and drosophila.

In some embodiments, the biological sample is a mammalian cell; in a specific embodiment, the biological sample is, for example, a human HEK293T cell.

In some embodiments, it is preferred that the site-directed introduction of the unnatural amino acid PrAK is in an orthogonal translation system using a variant of the pyrrolysine-tRNA synthetase activity from Methanosarcina mazei, Methanosarcina mazei, and cognate trnas thereof. More preferably, the pyrrolysine-tRNA synthetase active mutant is a Y306A, L309A and Y384F mutant PrAKRS based on wild-type pyrrolysine-tRNA synthetase of Methanosarcina mazei, and the amino acid sequence of the mutant is shown in SEQ ID NO. 1.

The invention also provides the pyrrolysinyl tRNA synthetase PrAKRS and a coding gene thereof, and the amino acid sequence of the pyrrolysinyl tRNA synthetase PrAKRS is shown in SEQ ID NO. 1.

The invention also provides a gene for encoding the pyrrolysinyl tRNA synthetase PrAKRS.

The invention also provides application of the pyrrolysinyl tRNA synthetase PrAKRS in fixed-point introduction of unnatural amino acid into biological macromolecules;

preferably, the unnatural amino acid is the aforementioned unnatural amino acid PrAK;

preferably, the biological macromolecule is a fluorescent protein or luciferase; more preferably, the fluorescent protein or luciferase is Enhanced Green Fluorescent Protein (EGFP) or firefly luciferase (ffluc).

The invention also provides an orthogonal translation system, wherein the orthogonal translation system is an E.coli cell comprising the aforementioned pyrrolysinyl tRNA synthetase PrAKRS and a corresponding cognate tRNA.

The present invention also provides a method for producing a protein comprising an unnatural amino acid at a selected position in an orthogonal translation system, which comprises introducing the unnatural amino acid using the aforementioned pyrrollysyl tRNA synthetase PrAKRS as an aminoacyl-tRNA synthetase, or introducing the unnatural amino acid using the aforementioned orthogonal translation system.

In some embodiments, the method introduces the aforementioned unnatural amino acid PrAK, and in particular, the method comprises co-expressing the aforementioned pyrrolysinyl-tRNA synthetase praksrs gene, cognate tRNA, and a fluorescent protein or luciferase gene in which the site at which the unnatural amino acid PrAK is to be introduced is mutated to the amber stop codon TAG in the presence of the unnatural amino acid PrAK in a biological sample, thereby expressing the fluorescent protein or luciferase protein introduced with PrAK at a specific site, i.e., a genetically encoded formaldehyde fluorescent probe or bioluminescent probe, in the biological sample.

In some embodiments, the biological sample is transformed into both the first and second expression vectors; the first expression vector contains a pyrrolysinyl-tRNA synthetase gene and a homologous correlation tRNA thereof, and the second expression vector contains a fluorescent protein or luciferase gene which is to be introduced with an unnatural amino acid PrAK and has a specific site mutated into an amber stop codon TAG. In a biological sample, a fluorescent protein or luciferase gene with a specific site mutated into an amber stop codon TAG is translated, and the pyrrollysyl-tRNA synthetase PrAKRS gene and homologous association tRNA thereof can specifically recognize the amber stop codon TAG, so that the unnatural amino acid PrAK site is specifically introduced into the fluorescent protein or luciferase protein. In a biological sample, the newly expressed pyrrolysinyl-tRNA synthetase prakrrs charges endogenous trnas with natural or unnatural amino acids at no significant level, or in some cases at no detectable level; on the other hand, endogenous aminoacyl-tRNA synthetases in a biological sample do not charge newly expressed cognate trnas for pyrrolysinyl-tRNA synthetases with natural or unnatural amino acids.

In some embodiments, the fluorescent protein is Enhanced Green Fluorescent Protein (EGFP), which has been specifically site-directed to the unnatural amino acid lysine analog PrAK at lysine position 85 by the orthogonal translation system described above. In some embodiments, the luciferase is a firefly luciferase (ffluc) to which the unnatural amino acid lysine analog PrAK as described above is specifically site-directed at the 529 th lysine site by the orthogonal translation system described above.

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

1) the genetically encoded formaldehyde-reactive unnatural amino acid PrAK prepared in the invention can be specifically inserted into biomacromolecule fluorescent proteins or bioluminescent proteins, and the fluorescence and bioluminescence of the biomacromolecule fluorescent proteins or bioluminescent proteins can be controlled. The unnatural amino acid PrAK-modified EGFP and ffluc mutants are not fluorescent and bioluminescent, respectively, when formaldehyde is not present; when formaldehyde exists, the unnatural amino acid PrAK reacts with the formaldehyde to recover key lysine residues, so that the fluorescence and bioluminescence of the amino acid PrAK are recovered, and the formaldehyde detection is realized.

2) The probe prepared based on PrAK can combine the 2-azacopopus rearrangement reaction activity of formaldehyde with site-specific protein engineering and regulation, and provides selective fluorescence and bioluminescence enhanced response for formaldehyde with biological correlation concentration rather than other potential interfering molecules;

3) the probes prepared by the invention can image formaldehyde in living cells, and because the probes have the characteristic of genetic coding, the probes provide a plurality of advantages for formaldehyde imaging, such as stable marking, the advantage of intracellular retention, easy cell-specific or organ-specific targeting operation, easy introduction of living tissues and the like;

4) the invention develops the pyrlysyl tRNA synthetase mutant PrAKRS (Y306A, L309A, Y384F), and can effectively introduce PrAK into macromolecular fluorescent or bioluminescent protein to prepare a reactive fluorescent probe and a bioluminescent probe;

5) the lysine analogue PrAK, the reaction type fluorescent probe and the bioluminescence probe thereof disclosed by the invention have the advantages of convenience, non-invasiveness, instantaneity, visualization and the like for detecting and imaging formaldehyde in a biological sample.

Drawings

FIG. 1: based on the genetic code formaldehyde introduced by PrAK fixed point (A) fluorescence probe and (B) bioluminescence probe principle and (C) PrAK structure and its 2-aza copple rearrangement reaction with formaldehyde;

FIG. 2: a PrAK synthetic route;

FIG. 3: Fmoc-PrAK synthetic route;

FIG. 4: LC-MS analysis of Fmoc-PrAK reaction with Formaldehyde: (A) Fmoc-PrAK, (B) a reaction mixture of Fmoc-PrAK and formaldehyde and (C) an HPLC profile of Fmoc-L-Lys at 254 nm; (D) mass spectrum of HPLC peak at 19.079 min shown in (a); (E) mass spectrum of HPLC peak at 18.365 min shown in (B); (F) mass spectrum of HPLC peak at 17.273 min shown in (B); (G) mass spectrum of HPLC peak at 17.225 min shown in (C);

FIG. 5: screening a result graph of inserting PrAK into EGFP-Y39TAG of the pyrrolysinyl tRNA synthetase mutant in escherichia coli by using a fluorescence spectrometry method;

FIG. 6: results of PrAK site-specific insertion into EGFP in e.coli and mammalian cells: (A) detecting Escherichia coli EGFP-Y39TAG by immunoblotting, inserting BocK and PrAK, and using Coomassie brilliant blue staining (CB) as a sample loading amount control; BocK is N (e) -Boc-L-lysine, which is a well-recognized excellent substrate for wild-type pyrrolysinyl tRNA synthetase (MmPylRS); (B) detecting the insertion of PrAK in Escherichia coli EGFP-K85TAG by immunoblotting, and taking Coomassie brilliant blue staining (CB) as a sample loading amount control; (C) detecting the EGFP-Y39TAG and EGFP-K85TAG insertion PrAK of HEK293T cells by immunoblotting;

FIG. 7: SDS-PAGE electrophoresis picture of EGFP-K85PrAK protein purified from escherichia coli;

FIG. 8: in-vitro fluorescence detection of formaldehyde by using purified EGFP-K85 PrAK: (A) fluorescent response of EGFP-K85PrAK (0.5. mu.M) to different concentrations of formaldehyde. (B) Relative fluorescence response of EGFP-K85PrAK (0.5 μ M) to relevant biomolecules (2mM) (λ em ═ 510 nm); EGFP-K85PrAK was incubated with formaldehyde or related biomolecules at 37 ℃ for 1 hour, and fluorescence signals were detected in PBS (20mM, pH7.4) with excitation wavelength of 468 nm;

FIG. 9: fluorogenic in vitro detection of formaldehyde for purified EGFP-K85 PrAK: (A) fluorescent response of EGFP-K85PrAK (0.5. mu.M) to different concentrations of formaldehyde; (B) linear relationship of EGFP-K85PrAK (0.5. mu.M) to formaldehyde fluorescence response; (C) fluorescent response of EGFP-K85PrAK (0.5. mu.M) to low concentrations of formaldehyde. (D) Kinetics of fluorescent response of EGFP-K85PrAK (0.5. mu.M) to 100. mu.M formaldehyde; fluorescence intensity was recorded at a maximum emission intensity of 510nm under 468nm excitation in 20mM PBS at room temperature; data are shown as mean fluorescence intensity ± standard deviation (n ═ 3); statistical analysis of multiple comparisons in (C) was performed using one-way analysis of variance in GraphPad Prism, # p < 0.01;

FIG. 10: ESI-MS validation of formaldehyde reacted with EGFP-K85PrAK FIG: (A) schematic diagram of molecular weight change of EGFP-K85PrAK after reaction with formaldehyde; (B) ESI-MS spectra of EGFP-K85PrAK proteins expressed in E.coli. The molecular weight peak at 30,307.78Da indicates a full-length EGFP-K85PrAK protein that does not form a chromophore; (C) ESI-MS spectrum of EGFP-K85PrAK protein after formaldehyde treatment; molecular weight peaks of 30,124.59Da and 29,993.23Da indicate that the formaldehyde reaction can generate native lysine residues; the molecular weight peak at 29,973.84Da indicates the formation of EGFP chromophore (-20 Da);

FIG. 11: fluorescence confocal imaging profile for formaldehyde detection in HEK293T living cells expressing EGFP-K85 PrAK: (A) with NaHSO₃(1mM) pretreatment of cells expressing EGFP-K85PrAK or EGFP-K85BocK followed by incubation with formaldehyde (1mM) for 1 hour prior to imaging; (B) EGFP-K85PrAK expressing cells were incubated with THFA (2mM) or 5,10-me-THFA (1mM) for 1 hour and allowed to fluoresceAn image; cell nuclei were stained with Hoechst 33342;

FIG. 12: EGFP-K85PrAK fluorescence detection of formaldehyde in live HEK293T cells: (A) fluorescence imaging of different concentrations of formaldehyde by EGFP-K85PrAK in living cells, treating HEK293T cells expressing EGFP-K85PrAK with different concentrations of formaldehyde in BSS buffer for 1h, and then imaging with a confocal fluorescence microscope; (B) comparison of mean fluorescence intensity with formaldehyde-treated HEK293T cells with respect to that without formaldehyde treatment showed data as mean fluorescence intensity ± standard deviation (n-3); statistical analysis was performed on two comparisons using the Student t-test in GraphPad Prism,. p < 0.0001;

FIG. 13: imaging graphs of formaldehyde generated by Tetrahydrofolate (THFA) metabolism in live HEK293T cells using EGFP-K85PrAK fluorescence: HEK293T cells expressing EGFP-K85PrAK were treated with different concentrations of THFA in BSS buffer for 1h, and then imaged with a confocal fluorescence microscope;

FIG. 14: SDS-PAGE EPE of expression and purification of fLuc-K529PrAK in E.coli: (A) western blot analysis of fLuc-K529BocK and fLuc-K529PrAK expression in E.coli: (B) SDS-PAGE gel analysis of purified fLuc-K529BocK and fLuc-K529PrAK proteins;

FIG. 15: bioluminescence assay detection of formaldehyde in solution and cells using fLuc-K529 PrAK: (A) bioluminescent response of purified fLuc-K529PrAK (0.5. mu.M) to different concentrations of formaldehyde; (B) quantification of bioluminescence intensity for the data shown in (a); (C) bioluminescence imaging of formaldehyde (0.5mM) in living cells with fLuc-K529 PrAK; (D) quantification of bioluminescence intensity of the data shown in (C);

FIG. 16: a graph of the results of bioluminescence assay using fLuc-K529PrAK for formaldehyde: (A) bioluminescent response of fLuc-K529PrAK (0.5. mu.M) to different concentrations of formaldehyde; (B) a linear relationship of the bioluminescent response of fLuc-K529PrAK (0.5. mu.M) to low concentrations of formaldehyde; (C) kinetics of bioluminescent response of fLuc-K529PrAK (0.5. mu.M) to 250. mu.M formaldehyde; (D) a luminescent response of fLuc-K529PrAK (0.5. mu.M) at a concentration of 500. mu.M to the relevant biological species; data were collected in 20mM PBS at room temperature; data are shown as mean intensity ± standard deviation (n ═ 3); statistical analysis of multiple comparisons in (B) was performed using one-way analysis of variance in GraphPad Prism, { p } 0.001;

FIG. 17: results of bioluminescence assay of formaldehyde in live HEK293T cells using fLuc-K529 PrAK: (A) bioluminescence imaging of different concentrations of formaldehyde in live HEK293T cells with fLuc-K529 PrAK; (B) bioluminescent response of fLuc-K529PrAK to low concentrations of formaldehyde in live HEK293T cells; data are shown as mean bioluminescence intensity ± standard deviation (n-3); statistical comparisons were performed using one-way analysis of variance in GraphPad Prism, # p < 0.0001.

Detailed Description

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 illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by manufacturers, and are all conventional products available on the market.

Definition of partial terms

Unless defined otherwise below, all technical and scientific terms used in the detailed description of the present invention are intended to have the same meaning as 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 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 in the following a certain group is defined to comprise at least a certain number of embodiments, this should also be understood as disclosing a group which preferably only consists of these embodiments.

Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun.

The term "about" in the present invention denotes an interval of accuracy that can be understood by a person skilled in the art, which still guarantees the technical effect of the feature in question. The term generally denotes 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 only to aid in understanding the present invention. These definitions should not be construed to have a scope less than understood by those skilled in the art.

The term "genetic code" as used herein means: living cells translate the genetic material information encoded in the DNA or mRNA sequence into proteins. In certain aspects, the term "encoding" is used to describe the process of DNA semi-conservative replication, in which one strand of a double-stranded DNA molecule is used as a template for the synthesis of a new complementary sister strand encoded by a DNA-dependent DNA polymerase. In another aspect, the term "encode" refers to any procedure by which information within one molecule is used to direct the synthesis of a second molecule that is chemically different from the first molecule. For example, a DNA molecule can encode an RNA molecule, e.g., by a transcription process involving a DNA-dependent RNA polymerase. In addition, the RNA molecule can encode a polypeptide, such as a translation process. When the term "encode" is used to describe the translation process, the term also refers to the triplet codon that encodes the amino acid. In certain aspects, an RNA molecule can also encode a DNA molecule, for example, by a reverse transcription process involving an RNA-dependent DNA polymerase. In another aspect, a DNA molecule may encode a polypeptide, where "encoding" is understood to include both transcriptional and translational processes.

The term "formaldehyde-reactive" as used herein means: has the property of specific chemical reaction with formaldehyde. Reaction is also a chemical term, which means a process of generating a new chemical substance by an interaction between two or more chemical substances, i.e., a chemical reaction.

Thus, the "genetically encoded formaldehyde-reactive unnatural amino acids" of the invention refer to: having the property of chemically reacting with formaldehyde and being capable of participating in genetically encoded unnatural amino acids.

The term "association" as used herein: the term "cognate" refers to components that work together, e.g., an orthogonal tRNA and an orthogonal aminoacyl-tRNA synthetase. These components may also be referred to as "complementary" to each other.

The term "homologous" as used herein means: in genetics, the concept of homology refers primarily to sequence homology, indicating that two or more protein or DNA sequences have identical ancestors. Homologous sequences are also likely to have similar functions. Two sequences are either homologous or not, and there is no notion of "homology". The homologous portions of the sequence are also referred to as conserved (conserved).

The term "cognate tRNA" as used herein refers to: homologous tRNAs that are homologous to and function in conjunction with the translation system of interest, it is to be understood that it is actually preferred that substantially any amino acid, whether natural or unnatural, can be incorporated into an extended polypeptide using a cognate tRNA of the invention and a translation system that reacts to a selector codon during translation.

The term "pyrrolysinyl-tRNA synthetase" as used herein refers to: an enzyme that catalyzes activation of pyrrolysine and covalently binds to the 3' end of the corresponding tRNA molecule. Pyrrolysine (Pyl) is found in the methylamine methyltransferase of methanogens and is the currently known amino acid of position 22 involved in protein biosynthesis. Unlike the standard amino acid, pyrrolysine (Pyl) is formed from the sense code for the stop codon UAG. Correspondingly, the methanogen also contains specific pyrrolysinyl-tRNA synthetase (PylRS) and pyrrolysine tRNA (tRNA)^Pyl) It has a special structure different from the classical tRNA. Methanogenic bacteria generate pyrrolysinyl-tRNA by direct and indirect ways^Pyl(Pyl-tRNA^Pyl) It can be used forThe specific structure on the mRNA and other mechanisms not yet discovered control the UAG encoding to stop codon or pyrrolysine. The wild-type pyrrilinyl-tRNA synthetase (PylRS) can be obtained, for example, from Methanosarcina mazei (Methanosarcina mazei), Methanosarcina pasteurianus (Methanosarcina barkeri), Methanosarcina acetogenis (Methanosarcina acetovorans) and the like, which are methanogenic archaea, but is not limited thereto.

The term "mutant pyrrolysinyl-tRNA synthetase" as used herein refers to: the mutant was prepared by introducing a mutation into wild-type pyrrolysinyl-tRNA synthetase (PylRS) by various methods. The types of lysine derivatives that can be activated by the wild-type PylRS are limited, and for example, the pyrrolysyl tRNA synthetase mutant prakrrs of the present invention belongs to a "mutant pyrrolysyl-tRNA synthetase", and even an unnatural amino acid PrAK that is inactive when the wild-type PylRS is used can be efficiently introduced into a protein by the mutant PylRS of the present invention.

The term "amber stop codon TAG" as used herein means: transcription of the protein gene DNA results in a tripartite polynucleotide, TAG, corresponding to the termination codon UAG on the mRNA. In the present invention, "TAG" and "UAG" refer to amber stop codons on the corresponding DNA and mRNA, respectively.

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 invention may use the unnatural amino acid PrAK.

The term "affinity purification" as used herein means: a method for purifying protein, which utilizes the principle of specific binding between biological molecules to separate and purify biological substances.

The term "translation system" as used herein refers to: amino acids are incorporated into the components of an extended polypeptide chain (protein). Components of a translation system can include, for example, ribosomes, trnas, synthetases, mrnas, and the like. The O-tRNA and/or O-RS 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, such as a bacterium (e.g., E.coli), or in a eukaryotic cell, such as a yeast, mammalian cell, plant cell, algal cell, fungal cell, insect cell, etc.

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

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

EXAMPLE 1 design and preparation of unnatural amino acid lysine analog PrAK

The initial idea of the present invention was to synthesize a non-natural amino acid with formaldehyde reactivity specifically inserted into biomacromolecule fluorescent and bioluminescent proteins to control their fluorescence and bioluminescence, respectively (FIGS. 1A, 1B). The unnatural amino acid-modified EGFP and ffluc mutants have no fluorescent and bioluminescent activity in the absence of formaldehyde; in the presence of formaldehyde, the unnatural amino acid reacts with formaldehyde to revert to a critical lysine residue, resulting in restoration of its fluorescent and bioluminescent activity (fig. 1A, 1B), thereby enabling formaldehyde detection. To achieve the above objectives, the present invention autonomously designs a formaldehyde-reactive lysine analogue PrAK, which is proof of concept by detection and imaging of formaldehyde in biological samples.

The invention designs and synthesizes a formaldehyde-reactive unnatural amino acid derived from lysine, which is named PrAK, and the specific structure is shown in figure 1C, wherein the PrAK comprises a high allylamine group which reacts with formaldehyde and an N-propyl substituent which can increase nucleophilicity. PrAK and formaldehyde are subjected to addition reaction and dehydration to generate imine positive ions, then 2-azacopropol rearrangement reaction is carried out to generate a new imine positive ion, and L-lysine is finally obtained through further hydrolysis and beta-elimination (figure 1C). Based on the reaction, the formaldehyde can be detected and imaged by using the fluorescent protein and the bioluminescent protein.

The specific preparation method of PrAK comprises the following steps (see fig. 2):

compound 2

And (4) synthesizing.

According to the literature (Padhi, B.; Reddy, D.S.; Mohapatra, D.K., 2, 6-trans-disubstituted tetrahydropyran derivatives gold catalyzed diastereoselective synthesis: use of the C1-C13 fragment for the synthesis of the double-stranded amides A and B, RSC Advances 2015, 5(117), 96758-₂SO₄Dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/hexane ═ 1: 10) to give compound 2(16.66g, 67% yield).¹H NMR(400MHz,CDCl₃)7.20(d,J＝8.5Hz,2H),6.81(d,J＝8.6Hz,2H),5.81–5.65(m,1H),5.03(d,J＝3.7Hz,1H),5.00(s,1H),4.37(s,2H),3.72(s,3H),3.54–3.44(m,2H),2.69–2.63(m,1H),2.55–2.41(m,2H),2.20–2.07(m,2H),1.71–1.59(m,2H),1.47–1.33(m,2H),0.85(t,J＝7.4Hz,3H)。¹³C NMR(101MHz,CDCl₃)159.10,135.65,130.58,129.13,117.12,113.68,72.53,67.71,55.11,54.94,49.00,38.72,33.96,23.36,11.80。HRMS：C₁₇H₂₈NO₂[M+H]⁺278.2120 (calculated), 278.2115 (measured).

Compound 3

And (4) synthesizing.

To a solution of compound 2(16.66g, 60mmol) in 100mL THF was added Boc anhydride (20.7mL, 90mmol) and the reaction was stirred at 25 deg.C for 8h, followed by TLC (10% EtOAc/hexanes) to show complete consumption of compound 2. The reaction mixture was saturated with Na₂CO₃Diluted and extracted with EtOAc. Incorporated by referenceThe organic layer was washed with Na₂SO₄Drying, filtration and concentration under reduced pressure gave the Boc protected crude product as a colorless oil which was redissolved in CH₂Cl₂(100mL) and used directly in the next step. To this solution were added water (10mL) and 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone (DDQ; 34g, 150mmol) portionwise at 0 ℃. The reaction mixture was stirred at 25 ℃ for 2h with saturated NaHCO₃Aqueous solution (200mL) quenched with water (100mL) and CH₂Cl₂(200mL) dilution. The resulting mixture was stirred vigorously for 2 hours. The layers were separated and the aqueous layer was treated with CH₂Cl₂(3 × 150mL) combined organic layers were washed with brine (200mL) and dried over anhydrous Na₂SO₄Dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/hexane ═ 2: 3) to give alcohol 3 as a colorless oil (13g, 84% yield in two steps).¹H NMR(300MHz,CDCl₃)5.74(ddt,J＝17.0,10.2,6.7Hz,1H),5.17–4.95(m,2H),4.41–4.19(m,1H),3.57–3.39(m,3H),3.05–2.77(m,2H),2.48–2.27(m,1H),2.27–2.07(m,1H),1.61–1.50(m,2H),1.47(s,9H),0.86(t,J＝7.4Hz,3H)。¹³C NMR(101MHz,CDCl₃)157.29,135.16,116.71,79.66,58.60,51.59,44.76,37.67,35.34,28.30,23.38,11.51。HRMS：C₁₄H₂₇NO₃Na[M+Na]⁺280.1889 (calculated), 280.1883 (measured).

Compound 4

And (4) synthesizing.

To compound 3(3.22g, 12.5mmol) in CH at 0 deg.C₂Cl₂To a solution (30mL) were added pyridine (1.35mL, 16.8mmol) and 4-nitrophenyl chloroformate (3.4g, 16.8 mmol). The reaction mixture was stirred at 25 ℃ for 10h and quenched with water. The layers were separated and the aqueous layer was treated with CH₂Cl₂(50mL) extraction. The combined organic layers were saturated with NH₄Aqueous Cl (3 × 30mL) and brine (50mL) were washed with anhydrous Na₂SO₄Dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/hexane 20: 1) to giveCompound 4(3g, 57% yield) as a colorless oil.¹H NMR(300MHz,CDCl₃)8.25(d,J＝9.0Hz,2H),7.34(d,J＝9.0Hz,2H),5.73(ddt,J＝17.1,10.1,7.0Hz,1H),5.10–5.01(m,2H),4.30–3.95(m,3H),3.12–2.82(m,2H),2.52–2.32(m,1H),2.27–2.20(m,1H),2.03–1.87(m,2H),1.66–1.49(m,2H),1.43(s,9H),0.86(t,J＝7.4Hz,3H)。¹³C NMR(75MHz,CDCl₃)155.67,152.51,145.44,135.07,135.03,125.36,122.01,121.87,117.42,100.12,79.47,66.84,53.10,38.00,31.83,31.39,28.53,23.55,11.71。HRMS：C₂₁H₃₀N₂O₇Na[M+Na]⁺445.1951 (calculated) 445.1946 (measured).

Compound 5

And (4) synthesizing.

To a solution of N α -Boc-L-Lys (348mg, 1.41mmol) in DMF (5mL) at 0 deg.C was added a solution of DIPEA (0.47mL, 0.61mmol), DMAP (23mg, 0.19mmol) and Compound 4(396mg, 0.94mmol) in DMF (5mL) the reaction mixture was stirred at 25 deg.C for 10h and quenched with water the layers were separated and CH was added to the aqueous layer₂Cl₂(50mL) extraction the combined organic layers were washed with 1N HCl (3 × 15mL) and brine (20mL) and dried over anhydrous Na₂SO₄Dried and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (5% MeOH/CH)₂Cl₂) Compound 5 was obtained as a white solid (317mg, 64% yield).¹HNMR(400MHz,CD₃OD)5.75(td,J＝17.0,7.0Hz,1H),5.13–4.98(m,2H),4.05–3.87(m,3H),3.36–3.33(m,1H),3.13–3.05(m,2H),3.05–2.93(m,2H),2.42–2.33(m,1H),2.31–2.13(m,1H),1.98–1.71(m,3H),1.71–1.60(m,1H),1.59–1.31(m,24H),0.88(t,J＝7.4Hz,3H)。¹³CNMR(75MHz,CD₃OD)174.97,162.45,157.70,156.84,156.29,135.41,118.28,116.32,79.90,79.41,79.17,78.26,61.68,53.54,40.16,38.01,37.46,31.21,29.24,27.58,22.89,10.71。HRMS：C₂₆H₄₈N₃O₈[M+H]⁺530.3441 (calculated), 530.3467 (measured).

PrAK

And (4) synthesizing.

To a solution of compound 5(317mg, 0.6mmol) was added 8mL of HCl in 1, 4-dioxane (4M). The reaction mixture was stirred at 25 ℃ for 2 h. The solvent was removed under reduced pressure to give PrAK as a white solid (185.3mg, 94% yield).¹HNMR(400MHz,CD₃OD)5.84(td,J＝16.9,7.1Hz,1H),5.33–5.25(m,2H),4.29–4.05(m,2H),3.97(t,J＝6.0Hz,1H),3.41–3.38(m,1H),3.18–3.08(m,2H),3.02–2.96(m,2H),2.57–2.51(m,2H),2.04–2.01(m,2H),1.98–1.88(m,2H),1.77–1.71(m,2H),1.63–1.49(m,3H),1.50–1.47(m,1H),1.03(t,J＝7.3Hz,3H)。¹³C NMR(75MHz,CD₃OD)170.66,157.39,131.81,119.61,60.57,55.10,52.66,46.73,40.01,34.33,29.90,29.45,29.09,21.98,19.54,10.22。HRMS：C₁₆H₃₂N₃O₄[M+H]⁺330.2393 (calculated), 330.2381 (measured).

EXAMPLE 2 in vitro Fmoc-PrAK reactivity experiments with Formaldehyde

1) Fmoc-PrAK was prepared by the following specific method steps (see FIG. 3):

compound 6

And (4) synthesizing.

To a solution of N α -Fmoc-L-Lys (190mg, 0.474mmol) in DMF (5mL) was added a solution of DIPEA (100. mu.L, 0.61mmol) and Compound 4(143mg, 0.34mmol) in DMF (5mL), the reaction mixture was stirred at 25 ℃ for 10h and quenched with water₂Cl₂(50mL) extraction the combined organic layers were washed with 1N HCl (3 × 15mL) and brine (20mL) and dried over anhydrous Na₂SO₄Dried and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (5% MeOH/CH)₂Cl₂) Compound 6 was obtained as a white solid (86mg, 30% yield).¹H NMR(300MHz,d⁶-DMSO)7.89(d,J＝7.3Hz,2H),7.73(d,J＝7.1Hz,2H),7.60(d,J＝7.8Hz,1H),7.40(dd,J＝14.1,6.8Hz,2H),7.33(dd,J＝14.1,6.8Hz,2H),7.09(s,1H),5.81–5.57(m,1H),5.05–5.00(m,2H),4.36–4.13(m,3H),3.94–3.80(m,4H),3.65–3.14(m,2H),3.01–2.86(m,4H),2.30–2.21(m,2H),1.94–1.51(m,4H),1.49–1.25(m,13H),0.79(t,J＝6.7Hz,3H)。¹³C NMR(75MHz,d⁶-DMSO)174.61,156.73,155.43,144.43,144.38,141.30,136.28,128.21,127.64,125.87,120.69,117.32,79.00,78.71,66.17,61.63,54.42,47.25,38.19,37.61,32.82,32.31,31.05,29.59,28.62,23.49,22.50,11.99。HRMS：C₃₆H₄₉N₃O₈Na[M+Na]⁺674.3417 (calculated), 674.3412 (measured).

And (3) synthesizing Fmoc-PrAK.

To a solution of compound 6(36mg, 0.055mmol) was added 3mL of HCl in 1, 4-dioxane (4M). The reaction mixture was stirred at 25 ℃ for 2 h. The solvent was removed under reduced pressure to give Fmoc-PrAK (25mg, 83% yield) as a white solid.¹HNMR(500MHz,CD₃OD)7.78(d,J＝7.5Hz,2H),7.66(t,J＝8.4Hz,2H),7.38(t,J＝7.4Hz,2H),7.30(t,J＝7.4Hz,2H),5.92–5.63(m,1H),5.31–5.18(m,2H),4.45–4.28(m,2H),4.28–4.14(m,2H),4.12–4.04(m,2H),3.35–3.32(m,1H),3.18–3.11(m,2H),3.01–2.89(m,2H),2.59–2.43(m,2H),2.08–1.91(m,2H),1.85–1.84(m,1H),1.75–1.68(m,3H),1.59–1.46(m,2H),1.45–1.33(m,2H),0.99(t,J＝7.4Hz,3H)。¹³C NMR(126MHz,CD₃OD)178.68,178.61,158.68,158.08,145.46,145.27,142.59,132.95,128.75,128.14,126.21,120.90,120.74,67.76,61.51,57.23,56.12,56.06,49.85,48.53,47.49,41.50,35.49,35.40,33.51,31.17,30.52,30.45,23.71,20.82,11.30。HRMS：C₃₁H₄₂N₃O₆[M+H]⁺552.3074 (calculated), 552.3066 (measured).

2) Reaction of Fmoc-PrAK with Formaldehyde

Formaldehyde solution (final 1mM) was added to Fmoc-PrAK (0.5mM) solution (PBS and acetonitrile mixed solvent (v/v ═ 1: 1) — the resulting mixture was incubated at 37 ℃ for 1 hour, then the reaction mixture was directly analyzed by LC-MS using Shimadzu LCMS-2020 system set to 254nm with absorption at 360nm as a reference.

The reaction of Fmoc-PrAK with formaldehyde was analyzed by LC-MS using liquid chromatography-mass spectrometry, and the results confirmed the production of Fmoc-Lys product and aldehyde intermediate (FIG. 4), thereby verifying that PrAK has 2-azacopopus rearrangement reactivity to formaldehyde, and is likely to be applied to formaldehyde detection.

Example 3 site-directed introduction of unnatural amino acid PrAK into proteins

1) PrAKRS sequence optimization and screening

To obtain active site mutants of pyrrolysinyl tRNA synthetases capable of inserting praks, the PrAK computational simulation was modeled into the pyrrolysine binding pocket of pyrrolysinyl tRNA synthetase, and it was noted that the side chain of PrAK may be in spatial conflict with many residues around the pocket (e.g., Y306, L309, C348, M350, I405, and I413V). Therefore, a series of active site mutants based on wild-type pyrrolysinyl tRNA synthetase were constructed by mutating these residues to less sterically hindered amino acids by rational design, as shown in the following table.

Mutant group	Mutation site
		Group
1	L309A,Y384F
		Group
2	L309A,C348S,Y384F
		Group
3	Y384F
		Group
4	Y306A,Y384F
		Group
5	Y306G,L309G,Y384F
		Group
6	L309A,Y348F,Y384F
		Group 7	Y306G,L309A,Y384F
Group
	8	Y306A,L309G,Y384F
Group 9			Y306A,L309A,Y384F
	Group
10		Y306A,L309A,C348A,Y384F
	Group 11		Y306A,L309A,Y384F,I405R
Group
	12	Y306A,L309A,C348S,Y384F
Group 13			Y306A,L309A,C348S,Y384F,I405R
	Group 14	Y306A,L309A,Y384F,I413V
Group
	15	Y306A,L309A,M350A,Y384F

In order to screen PrAKRS, the pLX-EGFP-Y39TAG plasmid and pBX-PylRS active site mutant plasmid are co-transformed into an Escherichia coli strain BL 21. The transformed monoclonal bacteria were cultured with shaking overnight at 37 ℃ in LB medium containing kanamycin (40. mu.g/mL) and chloramphenicol (34. mu.g/mL), followed by culturing at 1: 100 dilutions were inoculated into fresh TB medium (pH 8.0) supplemented with kanamycin (40. mu.g/mL) and chloramphenicol (34. mu.g/mL) and cultured with shaking at 37 ℃. When the OD600 reached 0.6, 2mM PrAK was added to the bacterial culture. After 1 hour of incubation, protein expression was induced by 1mM isopropyl-1-thio- β -D-galactopyranoside (IPTG) for 10 hours at 37 ℃. The fluorescence intensity of individual bacterial cultures was measured on a microplate reader and compared to control cultures without PrAK added.

It is desirable that a mutant of pyrrollysyl tRNA synthetase (Y306A, L309A, Y384F, named PrAKRS) with the sequence shown in SEQ ID NO.1 was found to efficiently introduce PrAK into EGFP-Y39TAG (FIG. 5).

The sequence of SEQ ID NO.1 is as follows:

MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMLAPNLANYARKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFCQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVFGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL。

2) incorporation of PrAK into proteins of E.coli

The desired plasmid containing the amber stop codon TAG was co-transformed with the pBX-PrAKRS plasmid into E.coli strain BL21(DE 3). The transformed bacteria were grown overnight at 37 ℃ in LB medium containing kanamycin (40. mu.g/mL) and chloramphenicol (34. mu.g/mL), and then cultured at a temperature of 1: 100 dilutions were inoculated into fresh TB medium (pH 8.0) supplemented with kanamycin (40. mu.g/mL) and chloramphenicol (34. mu.g/mL) and cultured with shaking at 37 ℃. When the OD600 reached 0.6, 2mM PrAK was added to the bacterial culture. After 1 hour of incubation, protein expression was induced by addition of 1mM isopropyl-1-thio- β -D-galactopyranoside (IPTG) for 10 hours. The cells were then harvested by centrifugation and lysed with 4% SDS lysis buffer (4% SDS, 150mM NaCl, 50mM triethanolamine, pH7.4) at 95 ℃. The resulting cell lysate was centrifuged at 16,000x g for 5 minutes at room temperature to remove cell debris. Protein concentration was determined by BCA assay. Finally, cell lysates were electrophoretically separated on home-made SDS-PAGE gels and analyzed by immunoblotting and coomassie blue staining.

The results show that full-length EGFP protein is expressed only when PrAK is present in the medium. Thus, PrAK successfully integrated into the Y39 and K85 sites of e.coli EGFP (fig. 6A and 6B). Also, PrAK showed insertion efficiency in e.coli comparable to BocK (fig. 6A). BocK is N (e) -Boc-L-lysine, which is a well-recognized excellent substrate for wild-type MmPhylRS.

3) Incorporation of PrAK into proteins of mammalian cells

HEK293T cells were seeded onto polylysine coated 12-well plates and cultured overnight in 1mL of growth medium. The next day, the objective plasmid containing the amber codon TAG (0.65. mu.g per well) and PylRS plasmid (0.35. mu.g per well) were co-transfected into cells using PEI (2.5. mu.g per well) with or without PrAK (1 mM). To express PrAK-modified proteins, pEF1 α -FLAG-prakrrs was used; pEF1 α -FLAG-MmPylRS was used on the other hand as a control spiked into BocK. 24 hours after transfection, cells were lysed with 4% SDS lysis buffer containing protease inhibitors and beta-mercaptoethanol by sonication and vortexing. The resulting cell lysate was centrifuged at 16,000x g for 5 minutes at room temperature to remove cell debris. Protein concentration was determined by BCA assay. Finally, cell lysates were separated on an in-house SDS-PAGE gel and analyzed by immunoblotting.

The results show that in mammalian HEK293T cells, full-length EGFP protein was expressed only when PrAK was present in the culture medium, indicating that the genetic encoding of Y39 and K85 site PrAK of EGFP was successfully achieved in mammalian HEK293T cells (fig. 6C).

Taken together, the results indicate that PrAK can be site-specifically encoded into EGFP by a PrAKRS mutant in both e.

Example 4 preparation of EGFP-K85PrAK and in vitro Formaldehyde assay for EGFP-K85PrAK

1) Preparation of EGFP-K85PrAK

For the purification of EGFP-K85PrAK, the pLX-EGFP-K85TAG-Twin-Strep-TAG plasmid was co-transformed with the pBX-PrAKRS plasmid into E.coli BL21(DE 3). The transformed bacteria were grown overnight at 37 ℃ in LB medium containing kanamycin (40. mu.g/mL) and chloramphenicol (34. mu.g/mL), and then cultured at a temperature of 1: 100 dilutions were inoculated into fresh TB medium (pH 8.0) supplemented with kanamycin (40. mu.g/mL) and chloramphenicol (34. mu.g/mL) and cultured at 37 ℃. When the OD600 reached 0.6, 2mM PrAK was added to the bacterial culture. After 1 hour of incubation, protein expression was induced by 1mM isopropyl-1-thio- β -D-galactopyranoside (IPTG) for 10 hours at 37 ℃. The cells were collected and lysed with a sonicator in buffer W (100mM Tris-HCl, 150mM NaCl, 1mM EDTA, pH 8.0) containing a protease inhibitor cocktail. After centrifugation, the precipitate was dissolved in PBS buffer (PH 8.0) containing 8M urea. EGFP-K85PrAK protein was then purified using Strep-Tactin XT Superflow resin and eluted with BXT buffer (W buffer containing 50mM biotin). Stepwise refolding of EGFP-K85PrAK protein was performed on the column using a linear urea gradient from 6M to 0M. The SDS-PAGE electrophoresis chart of the purified EGFP-K85PrAK protein is shown in FIG. 7.

2) EGFP-K85PrAK in vitro formaldehyde detection

EGFP-K85BocK or EGFP-K85PrAK protein (0.5. mu.M) in 20mM PBS (pH7.4) was treated with different concentrations of formaldehyde in PBS buffer at 37 ℃ for 40 min in 96-well optical-bottom black plates. Excess formaldehyde was then quenched by addition of 5mM DTT and 300mM Tris-HCl (pH7.4) and incubation for 2h at 37 ℃. The fluorescence intensity at 510nm under 468nm excitation was then measured for each well on a microplate reader.

The results indicate that EGFP-K85PrAK is virtually non-fluorescent (FIG. 8A), confirming that position K85 is critical for EGFP fluorescence. In contrast, as the formaldehyde concentration of the solution increased, there was a clear fluorescence response at the maximum emission peak of 510nm, showing a formaldehyde concentration dependence (fig. 8A and 9A). At low formaldehyde concentrations, there was a linear relationship between EGFP-K85PrAK fluorescence intensity and formaldehyde concentration (FIG. 9B). The limit of in vitro detection of formaldehyde was estimated to be 25 μ M (FIG. 9C). Kinetic analysis showed that the fluorescence response of EGFP-K85PrAK to formaldehyde reached substantial saturation around 1h (FIG. 9D).

For the selectivity assay, 0.5. mu.M EGFP-K85PrAK protein was treated with 2mM formaldehyde or other potentially interfering reactive species (2mM) for 40 min at 37 ℃.5mM DTT and 300mM Tris-HCl (pH7.4) were then added to each sample and incubation at 37 ℃ for 2h quenched excess reaction species. The fluorescence intensity at 510nm under 468nm excitation was then measured for each well with three biological replicates on a microplate reader.

The results indicate that the compounds are compared with the potentially interfering biologically active carbonyl compounds and the common oxidation and reduction molecules (such as H) in the cell₂O₂And glutathione), EGFP-K85PrAK has good selectivity for formaldehyde (fig. 8B).

3) LC-MS analysis of EGFP-K85PrAK in vitro Formaldehyde detection

For further verification, the invention analyzes the protein molecular weight of EGFP-K85PrAK before and after formaldehyde treatment: the molecular weight observed before formaldehyde treatment (30176.584Da) was consistent with the theoretical molecular weight of the full-length EGFP-K85PrAK protein without chromophore formation, while the molecular weight observed after formaldehyde treatment (29973.844Da) was consistent with the molecular weight of the wild-type EGFP with chromophore formation (FIG. 10), confirming that the reaction of PrAK with formaldehyde reduces native lysine residues to form the EGFP chromophore.

Example 5 detection of Formaldehyde in EGFP-K85PrAK live cells

HEK293T cells were seeded onto polylysine coated 12-well plates and cultured overnight in 1mL of growth medium the next day, cells were co-transfected with PEI (2.5. mu.g per well) plasmids pEF1 α -FLAG-PrAKRS (0.35. mu.g per well) and pCMV-EGFP-K85TAG (0.65. mu.g per well). cells were completely cultured in the presence of PrAK (1 mM). after 24 hours of incubation, the cell culture medium was changedFresh Opti-MEM medium without PrAK. At 37 deg.C, 5% CO₂After further 3 hours of incubation, formaldehyde in BSS buffer (136.9mM NaCl, 5.37mM KCl, 1.26mM CaCl) was added₂、0.81mM MgSO₄、0.44mM KH₂PO₄、0.335mM Na₂HPO₄10mM PIPES, pH 7.2) for 1 hour and in 2 hours 4 washes with Opti-MEM medium to remove excess formaldehyde.the cells were then stained with NucBlue viable cell stain, the cells were changed to FluoroBrite DMEM, and imaged on a Nikon A1R confocal fluorescence microscope for the NucBlue channel using a 405nm laser as excitation light and fluorescent signals collected between 425nm and 475nm for the EGFP channel using a 488nm laser as excitation light and fluorescent signals collected between 500nm and 550nm for the NaHSO T-K-85 TAG cotransfection in control experiments in the presence of BocK (0.25mM) of HEK293T cells with plasmids pEF1 α -FLAG-Mm-PylRS and pCMV-EGFP-K-85 TAG for the NaHSO₃Treatment of cells with 1mM NaHSO in BSS buffer₃Treated and incubated with formaldehyde for 1 hour. At least three fields per well were randomly selected for each fluorescence imaging experiment. The fluorescence intensity of each Image was quantified in Image J and grouped for statistical analysis. Statistical analysis of multiple comparisons was performed using one-way analysis of variance in GraphPad Prism.

The results indicate that EGFP-K85PrAK is capable of detecting and imaging biologically relevant concentrations of formaldehyde from 0.1mM to 1mM intracellularly (FIGS. 11A and 12). In addition, use NaHSO₃(Formaldehyde scavenger) pretreatment of cells significantly attenuated the intracellular formaldehyde-induced fluorescence signal of EGFP-K85PrAK (FIG. 11A).

To verify that EGFP-K85PrAK can be visualized as endogenous formaldehyde, cells expressing EGFP-K85PrAK are treated with Tetrahydrofolate (THFA) or 5, 10-methylenetetrahydrofolate (5,10-me-THFA), which can be converted to formaldehyde in the folate cycle. Indeed, a strong fluorescent signal was indeed observed in the THFA or 5,10-me-THFA treated groups (fig. 11B and fig. 13), indicating that the probe was able to detect endogenously produced formaldehyde.

Taken together, these results indicate that EGFP-K85PrAK is well suited for fluorescence imaging of physiological levels of formaldehyde in living cells.

Example 6 fLuc-K529PrAK preparation and validation of Formaldehyde Performance in vitro assay

1) Preparation of fLuc-K529PrAK

For purification of fLuc-K529PrAK, pLX-fLuc-K529TAG-Twin-Strep-TAG plasmid was co-transformed with pBX-PrAKRS plasmid into E.coli BL21(DE 3). The transformed bacterial cells were grown overnight at 37 ℃ in LB medium containing kanamycin (40. mu.g/mL) and chloramphenicol (34. mu.g/mL), and then cultured at a temperature of 1: 100 dilutions were inoculated into fresh TB medium (pH 8.0) supplemented with kanamycin (40. mu.g/mL) and chloramphenicol (34. mu.g/mL) and cultured at 37 ℃. When the OD600 reached 0.6, 2mM PrAK was added to the bacterial culture. After 1 hour of incubation, protein expression was induced by addition of 1mM IPTG for 10 hours at 30 ℃.

In performing the expression analysis experiment, cells were collected and the resulting cell lysate was centrifuged at 16,000x g for 5 minutes at room temperature to remove cell debris. Protein concentration was determined by BCA assay. Finally, cell lysates were separated on an in-house SDS-PAGE gel and analyzed by immunoblotting and coomassie blue staining.

In preparing purified proteins, the collected cells are lysed in buffer W (100mM Tris-HCl, 150mM NaCl, 1mM EDTA, pH 8.0) containing a protease inhibitor cocktail using a sonicator. After centrifugation, the supernatant was collected and the protein was purified using Strep-Tactin XT Superflow resin and eluted with BXT buffer (W buffer containing 50mM biotin) according to the instructions, and the protein buffer was replaced with PBS buffer by dialysis.

Expression of fLuc-K529PrAK protein in E.coli is shown in FIG. 14A. Full-length ffluc protein is expressed only when PrAK is present in the culture medium. Thus, PrAK was successfully integrated into the K529 site of e.coli ffluc. The SDS-PAGE of the purified fLuc-K529PrAK protein is shown in FIG. 14B. Meanwhile, the ffluc-K529 BocK protein was expressed in the presence of BocK and purified in a similar manner (fig. 14B).

2) fLuc-K529PrAK in vitro detection of formaldehyde

fLuc-K529BocK or fLuc-K529PrAK protein (0.5. mu.M) was treated with different concentrations of formaldehyde for 30 min at 37 ℃ in 96-well optical bottom black plates. 100mM Tris-HCl (pH7.4) was then added to each well to quench excess formaldehyde. Luciferase activity was measured on a microplate reader using the Bright-Lumi luciferase assay kit and performed in three biological replicates. Bioluminescence images were taken on a ChemiDoc imaging system using a chemiluminescence detection mode.

The results show that ffluc-K529 PrAK shows a strong dose-dependent bioluminescence response to formaldehyde in aqueous solution containing ATP and the substrate fluorescein (fig. 15A, 15B and fig. 16), whereas no bioluminescence change was observed for ffluc-K529 BocK (fig. 15A and 15B). Meanwhile, fLuc-K529PrAK was able to detect formaldehyde at physiological concentrations ranging from 0.1 to 2mM within 0.5 hours (FIGS. 15A, 15B and 16).

For the selectivity assay, 0.5. mu.M fLuc-K529PrAK protein was treated with 0.5mM formaldehyde or other species that might interfere with the reaction in PBS buffer (20mM PBS, pH7.4) at 37 ℃. 100mM Tris-HCl (pH7.4) was then added to each well to quench excess active reaction species. Luciferase activity was measured as described above with three biological replicates.

The results indicate that the activity of the enzyme is comparable to that of potentially interfering biologically active species and to oxidative and reductive molecules (such as H) that are common in cells₂O₂And glutathione), ffluc-K529 PrAK had good selectivity to formaldehyde (fig. 16D).

Example 7 detection of Formaldehyde in fLuc-K529PrAK Living cells

HEK293T cells were seeded onto polylysine coated 96-well optical bottom black plates and cultured overnight in 0.2mL growth medium. The following day, the plasmids pEF1 α -FLAG-PrAKRS (35 ng per well) and pCMV-fLuc-K529TAG (65 ng per well) were co-transfected with PEI (250 ng per well) in complete cell growth medium with or without 1mM PrAK addition. After 24 hours of incubation, the cell culture medium was changed to fresh Opti-MEM medium without PrAK. For time-dependent luminescence imaging of formaldehyde, cells were treated with 0.5mM formaldehyde in BSS buffer for the indicated times. For dose-dependent luminescence imaging, cells were treated with different concentrations of formaldehyde in BSS buffer for 1 hour. Luciferase activity was measured on a microplate reader using the Bright-Lumi luciferase assay kit. Bioluminescence images were taken on a ChemiDoc imaging system using a chemiluminescence detection mode.

The results show that in live HEK293T cells expressing ffluc-K529 PrAK, the formaldehyde-treated group luminescence signal was significantly enhanced and exhibited formaldehyde dose dependence (fig. 15C, 15D and fig. 17). Meanwhile, in live HEK293T cells expressing fLuc-K529BocK, no enhancement of the luminescence signal was observed in the formaldehyde-treated group (fig. 15C and 15D).

Taken together, these data indicate that fLuc-K529PrAK is a gene-encoded bioluminescent probe capable of detecting physiological levels of formaldehyde in vitro and on living cells.

In conclusion, the invention successfully develops the formaldehyde-reactive lysine analogue PrAK, and the fluorescence probe and the bioluminescence probe which are coded by two genes of EGFP-K85PrAK and Luc-K529PrAK and prepared based on the formaldehyde-reactive lysine analogue PrAK can be used for detecting and imaging formaldehyde in solution and living cells.

The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined by the scope of the appended claims.

SEQUENCE LISTING

<110> Shenzhen institute of university of Beijing

<120> genetically encoded formaldehyde-reactive unnatural amino acid, preparation method and application thereof

<130>2020

<160>1

<170>PatentIn version 3.5

<210>1

<211>454

<212>PRT

<213> Artificial sequence

<400>1

Met Asp Lys Lys Pro Leu Asn Thr Leu Ile Ser Ala Thr Gly Leu Trp

1 5 10 15

Met Ser Arg Thr Gly Thr Ile His Lys Ile Lys His His Glu Val Ser

20 25 30

Arg Ser Lys Ile Tyr Ile Glu Met Ala Cys Gly Asp His Leu Val Val

35 40 45

Asn Asn Ser Arg Ser Ser Arg Thr Ala Arg Ala Leu Arg His His Lys

50 55 60

Tyr Arg Lys Thr Cys Lys Arg Cys Arg Val Ser Asp Glu Asp Leu Asn

65 70 75 80

Lys Phe Leu Thr Lys Ala Asn Glu Asp Gln Thr Ser Val Lys Val Lys

85 90 95

Val Val Ser Ala Pro Thr Arg Thr Lys Lys Ala Met Pro Lys Ser Val

100 105 110

Ala Arg Ala Pro Lys Pro Leu Glu Asn Thr Glu Ala Ala Gln Ala Gln

115 120 125

Pro Ser Gly Ser Lys Phe Ser Pro Ala Ile Pro Val Ser Thr Gln Glu

130 135 140

Ser Val Ser Val Pro Ala Ser Val Ser Thr Ser Ile Ser Ser Ile Ser

145 150 155 160

Thr Gly Ala Thr Ala Ser Ala Leu Val Lys Gly Asn Thr Asn Pro Ile

165 170 175

Thr Ser Met Ser Ala Pro Val Gln Ala Ser Ala Pro Ala Leu Thr Lys

180 185 190

Ser Gln Thr Asp Arg Leu Glu Val Leu Leu Asn Pro Lys Asp Glu Ile

195 200 205

Ser Leu Asn Ser Gly Lys Pro Phe Arg Glu Leu Glu Ser Glu Leu Leu

210 215 220

Ser Arg Arg Lys Lys Asp Leu Gln Gln Ile Tyr Ala Glu Glu Arg Glu

225 230 235 240

Asn Tyr Leu Gly Lys Leu Glu Arg Glu Ile Thr Arg Phe Phe Val Asp

245 250 255

Arg Gly Phe Leu Glu Ile Lys Ser Pro Ile Leu Ile Pro Leu Glu Tyr

260 265 270

Ile Glu Arg Met Gly Ile Asp Asn Asp Thr Glu Leu Ser Lys Gln Ile

275 280 285

Phe Arg Val Asp Lys Asn Phe Cys Leu Arg Pro Met Leu Ala Pro Asn

290 295 300

Leu Ala Asn Tyr Ala Arg Lys Leu Asp Arg Ala Leu Pro Asp Pro Ile

305 310 315 320

Lys Ile Phe Glu Ile Gly Pro Cys Tyr Arg Lys Glu Ser Asp Gly Lys

325 330 335

Glu His Leu Glu Glu Phe Thr Met Leu Asn Phe Cys Gln Met Gly Ser

340 345 350

Gly Cys Thr Arg Glu Asn Leu Glu Ser Ile Ile Thr Asp Phe Leu Asn

355 360 365

His Leu Gly Ile Asp Phe Lys Ile Val Gly Asp Ser Cys Met Val Phe

370 375 380

Gly Asp Thr Leu Asp Val Met His Gly Asp Leu Glu Leu Ser Ser Ala

385 390 395 400

Val Val Gly Pro Ile Pro Leu Asp Arg Glu Trp Gly Ile Asp Lys Pro

405 410 415

Trp Ile Gly Ala Gly Phe Gly Leu Glu Arg Leu Leu Lys Val Lys His

420 425 430

Asp Phe Lys Asn Ile Lys Arg Ala Ala Arg Ser Glu Ser Tyr Tyr Asn

435 440 445

Gly Ile Ser Thr Asn Leu

450

Claims

1. The structural general formula of the unnatural amino acid is shown as follows:

wherein R is alkyl; preferably, R is methyl, ethyl, propyl or benzyl.

2. The unnatural amino acid of claim 1, wherein R is propyl and said unnatural amino acid is PrAK, having the formula:

3. the unnatural amino acid of claim 2, wherein said unnatural amino acid is a genetically encoded formaldehyde-reactive unnatural amino acid.

4. A method for producing the unnatural amino acid according to any one of claims 2 to 3, which comprises the steps of:

and 4) removing the amino protecting group of the carbamate compound to generate the unnatural amino acid.

5. The method for producing an unnatural amino acid according to claim 4, wherein in step 1), said hydroxy group is protected as a p-methoxybenzyl group; preferably, the amine protection in step 2) is tert-butyloxycarbonyl (Boc); more preferably, the activation in step 3) is activating alcoholic hydroxyl group by using p-nitrophenyl chloroformate, and the amine protecting group is tert-butyloxycarbonyl (Boc) or fluorenylmethyloxycarbonyl (Fmoc).

6. A compound linked to the unnatural amino acid of any one of claims 1 to 3.

7. A composition comprising the unnatural amino acid of any one of claims 1 to 3.

8. An expression system, translation system or cell comprising the unnatural amino acid of any one of claims 1 to 3.

9. A biomacromolecule probe, wherein the biomacromolecule probe is characterized in that the unnatural amino acid as claimed in any one of claims 1 to 3 is introduced into a specific site of a biomacromolecule; preferably, the biomacromolecule probe is a genetically encoded fluorescent protein probe or bioluminescent protein probe.

10. The biomacromolecule probe of claim 9, wherein the fluorescent or bioluminescent protein is Enhanced Green Fluorescent Protein (EGFP) or firefly luciferase (ffluc), respectively; preferably, one of the critical lysine sites of the Enhanced Green Fluorescent Protein (EGFP) or the firefly luciferase (ffluc) is specifically introduced with the unnatural amino acid PrAK; more preferably, the key lysine site of the Enhanced Green Fluorescent Protein (EGFP) is K85; the key lysine site of the firefly luciferase (fLuc) is K529, namely the genetically encoded formaldehyde fluorescent probe and bioluminescent probe are EGFP-K85PrAK and fLuc-K529 PrAK.

11. A composition comprising the biomacromolecule probe of any one of claims 9-10.

12. An expression system, translation system or cell comprising a biomacromolecule probe according to any one of claims 9 to 10.

13. Use of a biomacromolecule probe according to any one of claims 9 to 10, or a composition according to claim 9, or an expression system, translation system or cell according to claim 10, for imaging or detecting formaldehyde in a sample.

14. The use of claim 13, wherein the sample is a biological sample; preferably, the biological sample is a live laboratory animal, more preferably, a mouse, zebrafish, nematode or drosophila.

15. The use of claim 13, wherein the biological sample is a mammalian cell; preferably, the biological sample is human HEK293T cells.

16. The use of claim 13, wherein the sample is an in vitro or extracellular sample.

17. A method for producing a biomacromolecule probe, characterized in that the unnatural amino acid according to any one of claims 1 to 3 is introduced into a biomacromolecule at a site.

18. The method of claim 17, wherein the biomacromolecule probe is a genetically encoded formaldehyde fluorescent probe or bioluminescent probe, and the genetically encoded formaldehyde fluorescent probe or bioluminescent probe is prepared by introducing the unnatural amino acid of any one of claims 1 to 3 to the fluorescent protein or luciferase biomacromolecule at a site.

19. The method of claim 18, comprising co-expressing in the host cell a pyrrolysinyl-tRNA synthetase gene, a cognate tRNA, and a fluorescent protein or luciferase gene having a mutation at a site where the unnatural amino acid PrAK is introduced into the amber stop codon TAG, in the presence of the unnatural amino acid PrAK.

20. The method of claim 19, further comprising a purification step of obtaining genetically encoded formaldehyde fluorescent probes and bioluminescent probes by purification; preferably, a fluorescent protein or luciferase protein incorporating PrAK at a specific site is prepared by affinity purification; more preferably, by Ni²⁺Column or Strep-

The column performs affinity purification on the tag protein.

21. The method of any one of claims 19-20, wherein the host cell is an e.coli cell, and the first and second expression vectors are simultaneously transformed into e.coli; the first expression vector contains a pyrrolysinyl-tRNA synthetase gene and a homologous correlation tRNA thereof, and the second expression vector contains a fluorescent protein or luciferase gene which is to be introduced with an unnatural amino acid PrAK and has a specific site mutated into an amber stop codon TAG; preferably, the host cell is E.coli BL21(DE 3).

22. The method of claim 21, wherein the pyrrollysyl-tRNA synthetase amino acid sequence is set forth in SEQ ID No. 1; more preferably, the fluorescent protein is Enhanced Green Fluorescent Protein (EGFP) in which a pro-85 th lysine site is specifically site-directed by an orthogonal translation system into the unnatural amino acid of any one of claims 1 to 3; or the luciferase is a firefly luciferase (fLuc) in which the 529 th lysine site is specifically site-directed by an orthogonal translation system to the unnatural amino acid described in any one of claims 1 to 3.

23. The method for imaging or detecting formaldehyde in a biological sample with an unnatural amino acid as claimed in any of claims 1 to 3, comprising the steps of:

1) introducing the unnatural amino acid of any of claims 1-3 to a fluorescent protein or luciferase biomacromolecule at a site in a biological sample to produce genetically encoded formaldehyde fluorescent probes and bioluminescent probes; or genetically encoded formaldehyde fluorescent and bioluminescent probes prepared by the method of any one of claims 17 to 22;

2) detecting formaldehyde in the biological sample using the genetically encoded formaldehyde fluorescent probe and the bioluminescent probe.

24. The method of claim 23, wherein the biological sample is a live laboratory animal, preferably a mouse, zebrafish, nematode, or fruit fly; alternatively, the biological sample is a mammalian cell, preferably a human HEK293T cell.

25. A pyrrolysinyl tRNA synthetase PrAKRS has an amino acid sequence shown in SEQ ID NO. 1.

26. A gene encoding the pyrrollysyl tRNA synthetase prakrrs of claim 25.

27. Use of the pyrrolysyl tRNA synthetase, prakrrs, of claim 25 for site-directed introduction of an unnatural amino acid into a biological macromolecule; preferably, the unnatural amino acid is the unnatural amino acid of any one of claims 1 to 3; more preferably, the biomacromolecule is a fluorescent protein or luciferase.

28. An orthogonal translation system, wherein the orthogonal translation system is an E.coli cell comprising the pyrrollysyl tRNA synthetase PrAKRS of claim 25 and a corresponding cognate tRNA.

29. A method of producing a protein comprising an unnatural amino acid at a selected position in an orthogonal translation system, comprising introducing the unnatural amino acid using the pyrrollysyl tRNA synthetase, prakrrs, of claim 25 as an aminoacyl-tRNA synthetase, or introducing the unnatural amino acid using the orthogonal translation system of claim 28.