WO2021248770A1 - Genetically encoded formaldehyde reactive unnatural amino acid, preparation method and application thereof - Google Patents

Abstract

A genetically encoded formaldehyde reactive unnatural amino acid. Specifically, the present invention relates to a genetically encoded formaldehyde reactive unnatural amino acid PrAK, a preparation method and an application thereof in detection and imaging of formaldehyde. The genetically encoded formaldehyde reactive unnatural amino acid PrAK can specifically introduce sites into biomacromolecules such as a green fluorescent protein (EGFP) and firefly luciferase (fLuc) in the protein translation process, construct a reactive biomacromolecule formaldehyde fluorescent probe and a bioluminescent probe, and can effectively detect or image formaldehyde in a biospecimen.

Description

A genetically coded formaldehyde reactive unnatural amino acid, preparation method and application thereof Technical field

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

technical background

Formaldehyde is one of the simplest reactive carbonyl species (RCS). It is a well-known basic chemical raw material widely used in industry and biomedicine, and it is also a common environmental hazard and carcinogen. But what is less known is that in a series of biological processes in biological systems, including epigenetic regulation, one-carbon unit of metabolizable energy, and alcohol detoxification, formaldehyde can be produced. For example, lysine-specific demethylase (LSD) or histone demethylase (JmjC domain-containing hisstone demethylases, JHDM) can produce formaldehyde by-products by catalyzing the demethylation of histones; In one-carbon unit metabolism, semicarbazide-sensitive amine oxidase (SSAO) catalyzes the deamination of methylamine to generate formaldehyde; in addition, methanol can be used by alcohol dehydrogenase 1 ( alcohol dehydrogenase 1, ADH1) oxidizes to form formaldehyde. In addition to a variety of endogenous sources of formaldehyde, there are also corresponding molecular mechanisms in the organism to decompose formaldehyde, such as aldehyde dehydrogenase (aldehyde dehydrogenase 2, ALDH2) or alcohol dehydrogenase 3 (alcohol dehydrogenase 3, ADH3, also known as The effect of ADH5) oxidizes it to formic acid. Therefore, under physiological conditions, the concentration of formaldehyde in organisms will be maintained at a stable level. For example, the concentration of formaldehyde in blood or urine is about 0.1 mM. In the brain and cells, it is about 0.2-0.4mM. Physiological level of endogenous formaldehyde is an important one-carbon source, which can be used to produce important cell structural units and act as a signal molecule to mediate normal physiological processes. However, it has been reported that the abnormal increase in the concentration of endogenous formaldehyde in the body is related to the occurrence of a series of diseases, including cancer, Alzheimer's disease, stroke and diabetes. In fact, the concentration of formaldehyde in the urine of patients with moderate and severe dementia is more than three times that of the healthy control group, reaching 0.3-0.4mM; and it is estimated that the concentration of formaldehyde in cancer tissues can be as high as 0.8mM. However, the exact role of formaldehyde in the physiological and pathological processes is still poorly understood, which has prompted continuous efforts to develop new tools for the detection of formaldehyde in biological samples.

Although traditional detection methods including colorimetric analysis, chromatography, and radiological testing can be used to measure the level of formaldehyde in plasma and homogenized tissues, these methods require complicated sample processing procedures, which can damage the integrity of the sample and limit the The application of non-invasive and in-situ detection of formaldehyde in living biological samples is introduced. Optical probes, especially fluorescent probes and bioluminescent probes, have the advantages of non-invasiveness, real-time and spatiotemporal imaging capabilities, and have become a powerful alternative to formaldehyde detection. Therefore, the development of formaldehyde optical probes (including formaldehyde fluorescent probes and bioluminescence probes) is essential for detecting and imaging the content of formaldehyde in biological samples and revealing the role of formaldehyde under physiological and pathological conditions. In the past few years, a series of small molecule fluorescent probes and bioluminescence probes for formaldehyde have been developed. However, there are few reports about protein-based genetically encoded formaldehyde fluorescent probes and bioluminescent probes. Compared with small molecule optical probes, protein-based genetically encoded formaldehyde fluorescent probes and bioluminescent probes can be introduced into living cells or organisms in the form of DNA, and have unique advantages such as superior cell retention, cell specificity and Specific targeting of sub-organelles, and stable and durable marking. In view of this, the present invention discloses a genetically encoded formaldehyde-responsive unnatural amino acid PrAK, which is site-specifically introduced into proteins, such as enhanced green fluorescent protein (EGFP) and firefly luciferase (fLuc), respectively. To construct protein-based genetically encoded formaldehyde fluorescent probes and bioluminescence probes to selectively detect and image formaldehyde in biological samples.

The key to constructing genetically-encoded formaldehyde fluorescent probes and bioluminescent probes using the formaldehyde-reactive unnatural amino acid PrAK is to insert them into the protein site-specifically. Protein is usually composed of 20 kinds of natural amino acids. As a template for protein synthesis, the sequence of nucleotides (A, U, C, G) on mRNA determines the sequence of protein amino acids. Three nucleotides on mRNA form a triplet code, which determines an amino acid. The four nucleotides A, U, C, G encode a total of 64 triplet codes, of which 61 are used to encode 20 standard amino acids, and the other three: UAA, UAG, and UGA are usually stop codons to guide proteins Termination of translation. A set of "orthogonal translation system" has been developed in this field, which can site-specifically insert various unnatural amino acids into proteins in prokaryotic and eukaryotic organisms. This system relies on an exogenous orthogonal protein that recognizes the appropriate selector codon (such as the stop codon UAG, which corresponds to the TAG on the DNA) during polypeptide translation in vivo, and inserts the required unnatural amino acid into a specified position. Translation components. This system uses orthogonal tRNA (O-tRNA) that recognizes the selector codon (such as the stop codon UAG, which corresponds to the TAG on the DNA), and then the corresponding specific orthogonal aminoacyl-tRNA synthetase (O-RS) ) Load the O-tRNA with unnatural amino acids. These components do not cross-react with any endogenous tRNA, aminoacyl-tRNA synthetase (RS), natural amino acids, or codons encoding natural amino acids in the host organism (that is, it must be orthogonal). Using this exogenous, orthogonal tRNA-RS pairing can genetically encode a large number of unnatural amino acids with different structures.

Those skilled in the art are familiar with how to use an "orthogonal translation system" suitable for preparing proteins containing unnatural amino acids, as well as general methods for generating orthogonal translation systems. For example, see International Publication No. WO2002/086075, titled "Methods and Composition for the Production of Orthogonal tRNA-Aminoacyl-tRNA Synthetase Pairs"; WO2002 /085923, named "In Vivo Incorporation of Unnatural Amino Acids"; WO2004/094593, named "Expanding the Eukaryotic Genetic Code"; WO2005/019415, It is called "Orthogonal Translation Components for the Incorporation of Unnatural Amino Acids" (Orthogonal Translation Components for the Incorporation of Unnatural Amino Acids). For other discussions of orthogonal translation systems incorporating unnatural amino acids and their production and use methods, please refer to the following papers: Wang and Schultz, "Expanding the Genetic Code" (Expanding the Genetic Code), Chem. Commun. (Camb.) 1: 1-11 (2002); Wang and Schultz, "Expanding the Genetic Code" (Expanding the Genetic Code), Angewandte Chemie Int. Ed. 44(1): 34-66 (2005); Xie and Schultz, "Expanding the Genetic Code" (An Expanding Genetic Code), Methods 36(3): 227-238 (2005); Xie and Schultz, "Adding Amino Acids to the Genetic Repertoire", Curr. Opinion in Chemical Biology 9(6 ): 548-554 (2005); Wang et al., "Expanding the Genetic Code" (Expanding the Genetic Code), Annu. Rev. Biophys. Biomol. Struct. 35: 225-249 (2006); Xie and Schultz, "Protein "Chemical Toolkit-Expanded Genetic Code" (A Chemical Toolkit for Proteins-an Expanded Genetic Code), Nat. Rev. Mol. Cell Biol. 7(10): 775-782 (2006).

Summary of the invention

The purpose of the present invention is to provide an unnatural amino acid (a lysine analogue) capable of reacting with formaldehyde, and a method for constructing a protein-based genetically encoded formaldehyde fluorescent probe or a bioluminescence probe by using the unnatural amino acid (a lysine analog). The present invention envisages the synthesis of an unnatural amino acid with formaldehyde reactivity, which is specifically inserted into biological macromolecule fluorescent or bioluminescent proteins to control their fluorescence and bioluminescence respectively (Figures 1A and 1B). When formaldehyde is not present, the unnatural amino acid modified EGFP and fLuc mutants have no fluorescence and bioluminescence, respectively; when formaldehyde is present, the unnatural amino acid reacts with formaldehyde to recover to key lysine residues, leading to its fluorescence and bioluminescence Recovery (Figure 1A, 1B), so as to achieve formaldehyde detection.

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

The present invention provides a class of unnatural amino acid lysine analogues that can react with formaldehyde, the general structure of which is as follows:

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

Specifically, such an unnatural amino acid is a lysine analog containing L-lysine, a homoallylamine group that reacts with formaldehyde, and a nitrogen-substituted alkyl group. It undergoes addition reaction with formaldehyde and dehydration to generate imine cations, followed by 2-azacopp rearrangement reaction to generate a new imine cation, and then through further hydrolysis and β-elimination to finally obtain L-lysine acid.

The present invention provides a non-natural amino acid lysine analogue PrAK that can react with formaldehyde, characterized in that the structural formula of the non-natural amino acid PrAK is as follows:

Specifically, the unnatural amino acid PrAK is a lysine analogue that contains L-lysine, a homoallylamine group that reacts with formaldehyde, and a nitrogen-substituted propyl group. PrAK and formaldehyde undergo an addition reaction and dehydration to form imine cations, followed by 2-azacop rearrangement reaction to form a new imine cation, and then through further hydrolysis and β-elimination to finally obtain L-lysine Acid (as shown in Figure 1C).

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

Step 1) The addition reaction of hydroxyl-protected 3-hydroxypropionaldehyde with propylamine and allyl boronic acid pinacol ester to prepare homoallylamine;

Step 2) Homoallylamine protects the amine group and removes the hydroxyl protecting group to generate an alcohol compound;

Step 3) The alcohol compound is activated by the alcohol hydroxyl group, and is connected with the L-lysine protected by the amine group to form a carbamate compound;

Step 4) The carbamate compound removes the amine protecting group to generate the unnatural amino acid PrAK.

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

In some embodiments, the amine protecting group in step 2) is tert-butoxycarbonyl (Boc);

In some embodiments, the activation in step 3) is to use p-nitrophenyl chloroformate to activate the alcohol hydroxyl group, and the amine protecting group is tert-butoxycarbonyl (Boc) or fluorenylmethyloxycarbonyl (Fmoc).

Specifically, the addition reaction of p-methoxybenzyl-protected 3-hydroxypropionaldehyde with propylamine and allyl boronic acid pinacol ester produces homoallylamine, and then the amine group uses tert-butoxycarbonyl (Boc) Protect and remove the p-methoxybenzyl protecting group to generate alcohol. Immediately afterwards, the alcoholic hydroxyl group is activated with p-nitrophenyl chloroformate and reacts with Boc-protected L-lysine to form a carbamate compound. Finally, the Boc protecting group is removed under acidic conditions to generate the unnatural amino acid PrAK. The specific route is shown in Figure 2. Fluorenylmethyloxycarbonyl (Fmoc) protected PrAK (Fmoc-PrAK) can be prepared by the same synthetic route, as shown in Figure 3, that is, the alcohol activated by p-nitrophenyl chloroformate and the L-lysine protected by Fmoc The reaction is connected to generate carbamate compound, and the Boc protecting group is removed to prepare Fmoc-PrAK.

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

The present invention also provides a compound linked to the aforementioned unnatural amino acid lysine analogue PrAK.

The present invention also provides a composition comprising the above-mentioned unnatural amino acid lysine analogue PrAK.

The present invention also provides an expression system, translation system or cell containing the above-mentioned unnatural amino acid lysine analogue PrAK.

The present invention also provides a biological macromolecule probe, which is characterized in that the biological macromolecule probe introduces the above-mentioned unnatural amino acid lysine analogue PrAK at a specific site of the biological macromolecule.

In some embodiments, the biomacromolecule probes are genetically encoded formaldehyde fluorescent probes and bioluminescence probes, which are characterized in that they include those with the aforementioned unnatural amino acid lysine analogue PrAK introduced at a specific site. Biological macromolecules such as fluorescent protein and luciferase; preferably, the biological macromolecules such as fluorescent protein and luciferase are enhanced green fluorescent protein (EGFP) and firefly luciferase (fLuc);

In some embodiments, one of the key lysine sites of the enhanced green fluorescent protein (EGFP) or firefly luciferase (fLuc) is specifically introduced into the unnatural amino acid PrAK; preferably, the enhanced green fluorescent protein ( The key lysine site of EGFP) is K85; the key lysine site of the firefly luciferase (fLuc) is K529; more preferably, the genetically encoded formaldehyde fluorescent probe or bioluminescence probe is EGFP-K85PrAK and fLuc-K529PrAK.

The present invention also provides a composition containing the above-mentioned biological macromolecular probe.

The present invention also provides an expression system, translation system or cell containing the above-mentioned biological macromolecular probe.

The present invention also provides an application of the above-mentioned biological macromolecules or genetically encoded formaldehyde fluorescent probes or bioluminescent probes in sample imaging or detection of formaldehyde;

In some embodiments, the sample is a biological sample; preferably, the biological sample is a living laboratory animal, such as mice, zebrafish, nematodes, and fruit flies; or the biological sample is mammalian cells, such as human origin HEK293T cells;

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

The present invention also provides a method for preparing biomacromolecule probes from the above-mentioned unnatural amino acid lysine analogue PrAK, which is characterized in that: the above-mentioned unnatural amino acid lysine analogue PrAK is introduced into the biomacromolecule at a designated point to prepare a biological macromolecule. Molecular probes.

In some embodiments, the biological macromolecule is a genetically-encoded formaldehyde fluorescent probe or a bioluminescent probe, which is characterized in that the aforementioned unnatural amino acid lysine analogue PrAK is added to biological macromolecules such as fluorescent protein and luciferase. The molecules are introduced at designated locations to prepare genetically encoded formaldehyde fluorescent probes or bioluminescent probes.

Specifically, the method includes co-expression of the pyrrolysyl-tRNA synthetase gene, homologously associated tRNA, and site mutations requiring the introduction of the unnatural amino acid PrAK in the host cell in the presence of the unnatural amino acid PrAK Fluorescent protein or luciferase gene with amber stop codon TAG;

In some embodiments, it further includes a purification step to obtain genetically encoded formaldehyde fluorescent probes or bioluminescent probes through purification;

Preferably, the fluorescent protein or luciferase protein into which PrAK is introduced at a specific site is prepared by affinity purification;

柱对标签蛋白进行亲和纯化。 More preferably, through a Ni ²⁺ column well known in the art or

The column performs affinity purification of the tagged protein.

In some embodiments, the host cell is an E. coli cell, and the first and second expression vectors are transformed into E. coli at the same time; the first expression vector also contains the pyrrolysyl-tRNA synthetase gene and its homology Associated with tRNA, the second expression vector contains a fluorescent protein or luciferase gene whose specific site is mutated into an amber stop codon TAG to be introduced into the unnatural amino acid PrAK. In the host cell, the fluorescent protein or luciferase gene whose specific site is mutated to the amber stop codon TAG During the translation process, the pyrrolysyl-tRNA synthetase gene and its homologous associated tRNA can specifically recognize the amber stop codon TAG, thereby introducing the unnatural amino acid PrAK site specifically into the fluorescent protein or luciferase protein. In the host cell, the newly expressed pyrrolysyl-tRNA synthetase does not load the endogenous tRNA with natural or unnatural amino acids at a significant level or in some cases not at a detectable level; on the other hand, in the host cell The source aminoacyl-tRNA synthetase will not use natural or unnatural amino acids to load the newly expressed tRNA homologously associated with the pyrrolysyl-tRNA synthetase.

In a specific embodiment, the host cell is, for example, Escherichia coli BL21 (DE3).

In some embodiments, preferably, the site-specific introduction of the unnatural amino acid PrAK uses a pyrrolysine-tRNA synthetase (PylRS) active mutant from Methanosarcina mazei (Mm) and Orthogonal translation system constituted by its homologous associated tRNA. More preferably, the pyrrolysine-tRNA synthetase active mutant is Y306A, L309A, Y384F based on the wild-type pyrrolysyl-tRNA synthetase (MmPylRS) of Methanosarcina mazei The mutant is named PrAKRS, and its amino acid sequence is shown in SEQ ID NO.1.

In some embodiments, the fluorescent protein is enhanced green fluorescent protein (EGFP). Through the aforementioned "orthogonal translation system", the original lysine site at position 85 is specifically introduced into the above-mentioned unnatural amino acid lysine. Analog PrAK. In some embodiments, the luciferase is firefly luciferase (fLuc). Through the aforementioned "orthogonal translation system", the original 529th lysine site is specifically introduced into the above-mentioned unnatural amino acid lysine. The acid analogue PrAK.

The present invention also provides a method for imaging or detecting formaldehyde in biological samples with the aforementioned unnatural amino acid lysine analogue PrAK, characterized in that:

Including the following steps:

1) In a biological sample, the unnatural amino acid lysine analogue PrAK is introduced into biological macromolecules such as fluorescent protein and luciferase to express genetically encoded formaldehyde fluorescent probe or bioluminescent probe;

2) Use genetically encoded formaldehyde fluorescent probes or bioluminescent probes to detect formaldehyde in biological samples.

Specifically, the method includes co-expression of the pyrrolysyl-tRNA synthetase gene, homologously associated tRNA, and site mutations requiring the introduction of the unnatural amino acid PrAK in a biological sample in the presence of the unnatural amino acid PrAK It is the fluorescent protein or luciferase gene of the amber stop codon TAG, so that the fluorescent protein or luciferase protein introduced into PrAK at a specific site is expressed in the biological sample, that is, the genetically encoded formaldehyde fluorescent probe or bioluminescent probe.

In some embodiments, the biological sample is transformed into the first and second expression vectors at the same time; the first expression vector contains both the pyrrolysyl-tRNA synthetase gene and its homologous associated tRNA, and the second The expression vector contains the fluorescent protein or luciferase gene of the unnatural amino acid PrAK that is mutated into the amber stop codon TAG at a specific site to be introduced. In biological samples, the fluorescent protein or luciferase gene whose specific site is mutated to the amber stop codon TAG During the translation process, the pyrrolysyl-tRNA synthetase gene and its homologous associated tRNA can specifically recognize the amber stop codon TAG, thereby introducing the unnatural amino acid PrAK site specifically into the fluorescent protein or luciferase protein. In biological samples, the newly expressed pyrrolysyl-tRNA synthetase does not load endogenous tRNA with natural or unnatural amino acids at a significant level or in some cases not at a detectable level; on the other hand, in biological samples The endogenous aminoacyl-tRNA synthetase will not use natural or unnatural amino acids to load newly expressed tRNA homologous to the pyrrolysyl-tRNA synthetase.

In some embodiments, the biological sample is a living laboratory animal, such as mice, zebrafish, nematodes, and fruit flies.

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

In some embodiments, preferably, the site-directed introduction of the unnatural amino acid PrAK is composed of a pyrrolysine-tRNA synthetase active mutant from Methanosarcina mazei and its homologous associated tRNA. Orthogonal translation system. More preferably, the pyrrolysine-tRNA synthetase active mutant is Y306A, L309A, Y384F mutant PrAKRS based on the wild-type pyrrolysyl-tRNA synthetase of Methanosarcina mazei , Its amino acid sequence is shown in SEQ ID NO.1.

In some embodiments, the fluorescent protein is enhanced green fluorescent protein (EGFP). Through the aforementioned "orthogonal translation system", the original lysine site at position 85 is specifically introduced into the above-mentioned unnatural amino acid lysine. Analog PrAK. In some embodiments, the luciferase is firefly luciferase (fLuc). Through the aforementioned "orthogonal translation system", the original 529th lysine site is specifically introduced into the above-mentioned unnatural amino acid lysine. The acid analogue PrAK.

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

The present invention also provides a gene encoding the aforementioned pyrrolysyl tRNA synthetase PrAKRS.

The present invention also provides an application of the aforementioned pyrrolysyl tRNA synthetase PrAKRS in the site-specific introduction of unnatural amino acids into biological macromolecules;

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

Preferably, the biological macromolecule is fluorescent protein or luciferase; more preferably, the fluorescent protein or luciferase is enhanced green fluorescent protein (EGFP) or firefly luciferase (fLuc).

The present invention also provides an orthogonal translation system, characterized in that the orthogonal translation system is an Escherichia coli cell, and comprises the aforementioned pyrrolysyl tRNA synthetase PrAKRS and corresponding homologous associated tRNA.

The present invention also provides a method for producing a protein containing an unnatural amino acid at a selected position in an orthogonal translation system, which is characterized in that it comprises using the aforementioned pyrrolysyl tRNA synthetase PrAKRS as an aminoacyl-tRNA synthetase for non-natural amino acid synthesis. The introduction of natural amino acids, or the use of the aforementioned orthogonal translation system for the introduction of unnatural amino acids.

In some embodiments, the method introduces the aforementioned unnatural amino acid PrAK. Specifically, the method includes co-expressing the aforementioned pyrrolysyl-tRNA synthetase in a biological sample in the presence of the unnatural amino acid PrAK. The PrAKRS gene, homologous associated tRNA, and the site where the unnatural amino acid PrAK needs to be introduced are mutated to the fluorescent protein or luciferase gene with the amber stop codon TAG, so as to express the fluorescent protein or fluorescence of the PrAK introduced at a specific site in the biological sample Prime enzyme protein, that is, genetically encoded formaldehyde fluorescent probe or bioluminescent probe.

In some embodiments, the biological sample is transformed into the first and second expression vectors at the same time; the first expression vector contains both the pyrrolysyl-tRNA synthetase gene and its homologous associated tRNA, and the second The expression vector contains the fluorescent protein or luciferase gene of the unnatural amino acid PrAK that is mutated into the amber stop codon TAG at a specific site to be introduced. In biological samples, the fluorescent protein or luciferase gene whose specific site is mutated to the amber stop codon TAG During the translation process, the pyrrolysyl-tRNA synthetase PrAKRS gene and its homologous associated tRNA can specifically recognize the amber stop codon Sub-TAG, thereby introducing the unnatural amino acid PrAK site specifically into the fluorescent protein or luciferase protein. In biological samples, the newly expressed pyrrolysyl-tRNA synthetase PrAKRS does not load endogenous tRNAs with natural or unnatural amino acids at significant levels or in some cases not at detectable levels; on the other hand, biological samples The mid-endogenous aminoacyl-tRNA synthetase will not use natural or unnatural amino acids to load newly expressed homologously associated tRNA with pyrrolysyl-tRNA synthetase.

In some embodiments, the biological sample is a living laboratory animal, such as mice, zebrafish, nematodes, and fruit flies.

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

In some embodiments, preferably, the site-directed introduction of the unnatural amino acid PrAK is composed of a pyrrolysine-tRNA synthetase active mutant from Methanosarcina mazei and its homologous associated tRNA. Orthogonal translation system. More preferably, the pyrrolysine-tRNA synthetase active mutant is Y306A, L309A, Y384F mutant PrAKRS based on the wild-type pyrrolysyl-tRNA synthetase of Methanosarcina mazei , Its amino acid sequence is shown in SEQ ID NO.1.

In some embodiments, the fluorescent protein is enhanced green fluorescent protein (EGFP). Through the aforementioned orthogonal translation system, the original lysine position at position 85 is specifically introduced into the above-mentioned unnatural amino acid lysine analogue. PrAK. In some embodiments, the luciferase is firefly luciferase (fLuc). Through the aforementioned orthogonal translation system, the original lysine position at position 529 is specifically introduced into the above-mentioned unnatural amino acid lysine.物PrAK.

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

1) The genetically encoded formaldehyde-reactive unnatural amino acid PrAK prepared by the present invention can be specifically inserted into biological macromolecular fluorescent proteins or bioluminescent proteins, and can control their fluorescence and bioluminescence. When formaldehyde is not present, the EGFP and fLuc mutants modified by the unnatural amino acid PrAK have no fluorescence and bioluminescence, respectively; when formaldehyde is present, the unnatural amino acid PrAK reacts with formaldehyde to recover to key lysine residues, causing its fluorescence and Bioluminescence is restored, and formaldehyde detection is realized.

2) The probe prepared based on PrAK of the present invention can combine the 2-azakop rearrangement reaction activity of formaldehyde with site-specific protein engineering and regulation, providing a biologically relevant concentration of formaldehyde instead of other potential interference molecules. Selective fluorescence and bioluminescence enhancement response;

3) The probes prepared by the present invention can image formaldehyde in living cells. Because these probes have the characteristics of heritable coding, they provide many advantages for formaldehyde imaging, such as stable labeling, advantages of intracellular retention, ease of cell specificity or Organ-specific targeting operations, and easy introduction into living tissues, etc.;

4) The present invention has developed the pyrrolysyl tRNA synthetase mutant PrAKRS (Y306A, L309A, Y384F), which can effectively introduce PrAK into macromolecular fluorescent or bioluminescent proteins to prepare reactive fluorescent probes and bioluminescent probes ；

5) The lysine analogue PrAK and its reactive fluorescent probes and bioluminescence probes of the present invention have many advantages such as convenience, non-invasiveness, real-time and visibility for the detection and imaging of formaldehyde in biological samples.

Description of the drawings

Figure 1: The principle of genetically encoded formaldehyde (A) fluorescent probe and (B) bioluminescence probe based on PrAK site-specific introduction, and (C) the structure of PrAK and its 2-azakop rearrangement reaction with formaldehyde;

Figure 2: PrAK synthetic route;

Figure 3: Fmoc-PrAK synthetic route;

Figure 4: LC-MS analysis of the reaction between Fmoc-PrAK and formaldehyde: (A) Fmoc-PrAK, (B) Fmoc-PrAK and formaldehyde reaction mixture, and (C) Fmoc-L-Lys HPLC profile at 254nm; ( D) The mass spectrum of the HPLC peak at 19.079 minutes shown in (A); (E) the mass spectrum of the HPLC peak at 18.365 minutes shown in (B); (F) The mass spectrum of the HPLC peak shown at 17.273 minutes in (B) The mass spectrum of the HPLC peak; (G) the mass spectrum of the HPLC peak at 17.225 minutes shown in (C);

Figure 5: Screening of pyrrolysyl tRNA synthetase mutants by fluorescence spectroscopy and inserting PrAK into EGFP-Y39TAG in E. coli;

Figure 6: Results of PrAK site-specific insertion into EGFP in E. coli and mammalian cells: (A) Western blot detection of EGFP-Y39TAG insertion of BocK and PrAK in E. coli EGFP-Y39TAG, using Coomassie Brilliant Blue (CB) as the top Sample size control; BocK is N(e)-Boc-L-lysine, which is recognized as an excellent substrate for wild-type pyrrolysyl tRNA synthetase (MmPylRS); (B) Western blot detection of E. coli EGFP-K85TAG Insert PrAK into PrAK, and use Coomassie Brilliant Blue (CB) as loading control; (C) Western blot detection of HEK293T cells EGFP-Y39TAG and EGFP-K85TAG inserted into PrAK;

Figure 7: SDS-PAGE electrophoresis diagram of EGFP-K85PrAK protein purified in E. coli;

Figure 8: In vitro fluorescence detection of formaldehyde with purified EGFP-K85PrAK: (A) Fluorescence response of EGFP-K85PrAK (0.5μM) to different concentrations of formaldehyde. (B) The relative fluorescence response (λem=510nm) of EGFP-K85PrAK (0.5μM) to related biomolecules (2mM); EGFP-K85PrAK and formaldehyde or related biomolecules were incubated at 37°C for 1 hour, in PBS (20mM, pH 7.4) The fluorescence signal is detected in 7.4), the excitation wavelength is 468nm;

Figure 9: Fluorescence in vitro detection of formaldehyde with purified EGFP-K85PrAK: (A) the fluorescence response of EGFP-K85PrAK (0.5μM) to different concentrations of formaldehyde; (B) the linearity of the fluorescence response of EGFP-K85PrAK (0.5μM) to formaldehyde Relationship: (C) Fluorescence response of EGFP-K85PrAK (0.5μM) to low concentration of formaldehyde. (D) Fluorescence response kinetics of EGFP-K85PrAK (0.5μM) to 100μM formaldehyde; under excitation at 468nm in 20mM PBS at room temperature, the fluorescence intensity was recorded at the maximum emission intensity of 510nm; the data shows the average fluorescence intensity ± standard deviation ( n=3); Use the one-way analysis of variance in GraphPad Prism to perform statistical analysis of multiple comparisons in (C), **p＜0.01;

Figure 10: ESI-MS verification diagram of the reaction between formaldehyde and EGFP-K85PrAK: (A) Schematic diagram of the molecular weight change of EGFP-K85PrAK after reaction with formaldehyde; (B) ESI-MS spectrum of EGFP-K85PrAK protein expressed in E. coli. The molecular weight peaks of 30,307.78 Da indicate that the full-length EGFP-K85PrAK protein does not form a chromophore; (C) the ESI-MS spectrum of the EGFP-K85PrAK protein after formaldehyde treatment; the molecular weight peaks of 30,124.59 Da and 29,993.23 Da indicate that the formaldehyde reaction can generate natural lysine Amino acid residue; the molecular weight peak of 29,973.84Da indicates the formation of EGFP chromophore (-20Da);

Figure 11: Confocal fluorescence imaging of detecting formaldehyde in HEK293T living cells expressing EGFP-K85PrAK: (A) Pre-treating cells expressing EGFP-K85PrAK or EGFP-K85BocK _{with NaHSO 3 (1mM), and then combining with formaldehyde before imaging} (1mM) incubate for 1 hour; (B) incubate cells expressing EGFP-K85PrAK with THFA (2mM) or 5,10-me-THFA (1mM) for 1 hour, and perform fluorescence imaging; cell nuclei are stained with Hoechst 33342;

Figure 12: EGFP-K85PrAK fluorescence detection of formaldehyde in live HEK293T cells: (A) EGFP-K85PrAK fluorescence imaging of different concentrations of formaldehyde in live cells, HEK293T cells expressing EGFP-K85PrAK in BSS buffer with different concentrations Treated with formaldehyde for 1 h, and then imaged with a confocal fluorescence microscope; (B) Compared with HEK293T cells that have not been treated with formaldehyde, the average fluorescence intensity of formaldehyde treatment is compared, and the data shows the average fluorescence intensity ± standard deviation (n=3); Use the Student t test in GraphPad Prism to perform statistical analysis on the two comparisons, ****p＜0.0001;

Figure 13: Using EGFP-K85PrAK to detect the formaldehyde produced by the metabolism of tetrahydrofolate (THFA) in live HEK293T cells: the HEK293T cells expressing EGFP-K85PrAK were treated with different concentrations of THFA in BSS buffer for 1 h, and then co-exposed Focused fluorescence microscope imaging;

Figure 14: Expression and purification of fLuc-K529 PrAK in E. coli SDS-PAGE electrophoresis diagram: (A) Western blot analysis of fLuc-K529BocK and fLuc-K529 PrAK expression in E. coli: (B) Purified fLuc-K529BocK And SDS-PAGE gel analysis of fLuc-K529PrAK protein;

Figure 15: Using fLuc-K529PrAK to perform bioluminescence analysis and detection of formaldehyde in solution and cells: (A) The bioluminescence response of purified fLuc-K529PrAK (0.5μM) to different concentrations of formaldehyde; (B)(A) Bioluminescence intensity quantification of the data; (C) fLuc-K529PrAK bioluminescence imaging of formaldehyde (0.5mM) in living cells; (D) quantification of the bioluminescence intensity of the data shown in (C);

Figure 16: Using fLuc-K529PrAK to detect formaldehyde bioluminescence results: (A) the bioluminescence response of fLuc-K529PrAK (0.5μM) to different concentrations of formaldehyde; (B) the bioluminescence response of fLuc-K529PrAK (0.5μM) to low concentrations of formaldehyde The linear relationship of the bioluminescence response; (C) the kinetics of the bioluminescence response of fLuc-K529PrAK (0.5 μM) to 250 μM formaldehyde; (D) the luminescence response of fLuc-K529PrAK (0.5 μM) to related biological species at a concentration of 500 μM; Collect data in 20mM PBS at room temperature; data are shown as average intensity ± standard deviation (n=3); use one-way analysis of variance in GraphPad Prism to perform statistical analysis of multiple comparisons in (B), ***p＜0.001 ；

Figure 17: Using fLuc-K529PrAK to perform bioluminescence detection results of formaldehyde in live HEK293T cells: (A) fLuc-K529PrAK bioluminescence imaging of different concentrations of formaldehyde in live HEK293T cells; (B) fLuc-K529PrAK on live HEK293T cells The bioluminescence response of medium and low concentrations of formaldehyde; the data is shown as the average bioluminescence intensity ± standard deviation (n=3); using the one-way analysis of variance in GraphPad Prism for statistical comparison, ****p<0.0001.

detailed description

The embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If specific conditions are not indicated in the examples, it shall be carried out in accordance with the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used without the manufacturer's indication are all conventional products that can be purchased on the market.

Definition of some terms

Unless otherwise defined below, the meanings of all technical and scientific terms used in the specific embodiments of the present invention are intended to be the same as those commonly understood by those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present invention.

As used in the present invention, the terms "including", "including", "having", "containing" or "involving" are inclusive or open-ended, and do not exclude other unlisted elements or method steps . The term "consisting of" is considered a preferred embodiment of the term "comprising". If in the following a certain group is defined as comprising at least a certain number of embodiments, this should also be understood as revealing a group preferably consisting only of these embodiments.

The indefinite or definite article used when referring to a noun in the singular form such as "a" or "an", "the" includes the plural form of the noun.

The term "approximately" in the present invention refers to an accuracy interval that can be understood by those skilled in the art and can still guarantee the technical effect of discussing the feature. The term usually means a deviation of ±10% from the indicated value, preferably ±5%.

In addition, the terms first, second, third, (a), (b), (c) and the like in the specification and claims are used to distinguish similar elements, and are not necessary for the order of description or time. It should be understood that the terms so applied are interchangeable under appropriate circumstances, and the embodiments described in the present invention can be implemented in other orders than described or exemplified in the present invention.

The following terms or definitions are only provided to help understand the present invention. These definitions should not be understood as having a scope less than that understood by those skilled in the art.

The term "genetic coding" used in the present invention refers to the translation of genetic material information encoded in DNA or mRNA sequences into proteins by living cells. In some aspects, the term "encoding" is used to describe the process of semi-conservative DNA replication, in which one strand of a double-stranded DNA molecule is used as a template, and a new complementary sister strand is encoded by a DNA-dependent DNA polymerase to synthesize. On the other hand, the term "encoding" refers to any procedure by which information within a molecule is used to guide the synthesis of a second molecule that is chemically different from the first molecule. For example, a DNA molecule can encode an RNA molecule, for example, through a transcription process involving DNA-dependent RNA polymerase. In addition, RNA molecules can encode polypeptides, such as during translation. When the term "encoding" is used to describe the translation process, the term also refers to the triplet codons that encode amino acids. In some aspects, RNA molecules can also encode DNA molecules, for example, through a reverse transcription process involving RNA-dependent DNA polymerase. On the other hand, a DNA molecule can encode a polypeptide. In this case, "encoding" should be understood to include both the transcription process and the translation process.

The term "formaldehyde reactivity" used in the present invention refers to the property of having a specific chemical reaction with formaldehyde. Reaction is also a chemical term, which refers to the process of generating new chemical substances by the interaction between two or more chemical substances, that is, chemical reaction.

Therefore, the "genetically encoded formaldehyde-reactive unnatural amino acid" in the present invention refers to an unnatural amino acid that has the property of chemically reacting with formaldehyde and can participate in genetic encoding.

The term "association" used in the present invention: the term "association" refers to components that work together, such as orthogonal tRNA and orthogonal aminoacyl-tRNA synthetase. These components can also be referred to as "complementary" to each other.

The term "homologous" as used in the present invention refers to: in genetics, the concept of homology mainly refers 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. Two sequences are either homologous or of different origin, and there is no concept of "homology". The homologous part of the sequence is also called conserved.

The term "homologously associated tRNA" used in the present invention refers to a homologous tRNA that is homologous to the translation system of interest and functions together. The selector codon reacts and essentially any amino acid (whether natural or unnatural) can be incorporated into the extended polypeptide.

The term "pyrrolysyl-tRNA synthetase" used in the present invention refers to an enzyme that catalyzes the activation of pyrrolysine and covalently binds to the 3'end of the corresponding tRNA molecule. Pyrolysine (Pyl) is found in the methylamine methyltransferase of methanogens and is currently the 22nd known amino acid involved in protein biosynthesis. Unlike standard amino acids, pyrrolysine (Pyl) is formed by the sense code of the stop codon UAG. Correspondingly, methanogens also contain specific pyrrolysyl-tRNA synthetase (PylRS) and pyrrolysine tRNA (tRNA ^Pyl ), which have a special structure different from classic tRNA. Methanogens produce pyrrolysyl-tRNA ^Pyl (Pyl-tRNA ^Pyl ) through direct and indirect pathways. It may control the UAG code to become a stop codon or pyrrolyl through a special structure on the mRNA and other undiscovered mechanisms. Acid. Wild-type pyrrolysyl-tRNA synthetase (PylRS), for example, can be obtained from Methanosarcina mazei, Methanosarcina barkeri, and Methanosarcina barkeri, which are methanogenic archaea. Cocci (Methanosarcina acetivorans), etc., but not limited to these.

The term "mutant pyrrolysyl-tRNA synthetase" used in the present invention refers to the production based on the wild-type pyrrolysyl-tRNA synthetase (PylRS) by introducing mutations in various methods. The types of lysine derivatives that can be activated by wild-type PylRS are limited. For example, the pyrrolysyl tRNA synthetase mutant PrAKRS in the present invention belongs to a "mutant pyrrolysyl-tRNA synthetase". The mutant PylRS can efficiently introduce protein even to the unnatural amino acid PrAK, which is inactive when wild-type PylRS is used.

The term "amber stop codon TAG" used in the present invention refers to the triple polynucleotide corresponding to the stop codon UAG on the mRNA formed by transcription on the protein gene DNA, namely TAG. In the present invention, "TAG" and "UAG" refer to the amber stop codons on the corresponding DNA and mRNA, respectively.

The term "unnatural amino acid" used in the present invention refers to any amino acid, modified amino acid and/or amino acid analogue that is not among the 20 common natural amino acids. For example, the present invention can use the unnatural amino acid PrAK.

The term "affinity purification" used in the present invention refers to a method of purifying protein, which uses the principle of specific binding between biological molecules to separate and purify biological substances.

The term "translation system" as used in the present invention refers to the incorporation of amino acids into each component of the elongating polypeptide chain (protein). The components of the translation system may include, for example, ribosomes, tRNA, synthetase, mRNA, and the like. The O-tRNA and/or O-RS of the present invention may be added to or part of an in vitro or in vivo translation system, for example, in non-eukaryotic cells, such as bacteria (such as E. coli), or in eukaryotic cells, such as Yeast, mammalian cells, plant cells, algae cells, fungal cells, insect cells, etc.

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

The present invention is further described by the accompanying drawings and the following examples. The accompanying drawings and examples are only used to illustrate specific embodiments of the present invention and should not be understood as limiting the scope of the present invention in any way.

Example 1 Design and preparation of unnatural amino acid lysine analogue PrAK

The original idea of the present invention is to synthesize an unnatural amino acid with formaldehyde reactivity and specifically insert it into biological macromolecule fluorescent and bioluminescent proteins to control their fluorescence and bioluminescence respectively (Figure 1A, 1B). When formaldehyde is not present, the EGFP and fLuc mutants modified with unnatural amino acids have no fluorescence and bioluminescence activity; when formaldehyde is present, the unnatural amino acid reacts with formaldehyde to recover to key lysine residues, resulting in fluorescence and bioluminescence. The luminescence activity is restored (Figure 1A, 1B), thereby realizing formaldehyde detection. In order to achieve the above objective, the present invention independently designs a formaldehyde-reactive lysine analogue PrAK, which is proved by the detection and imaging of formaldehyde in biological samples.

The present invention designs and synthesizes a formaldehyde-reactive unnatural amino acid derived from lysine, named PrAK. The specific structure is shown in Figure 1C. PrAK contains a high allylamine group that reacts with formaldehyde and a formaldehyde-reactive non-natural amino acid. N-propyl substituent. PrAK and formaldehyde undergo an addition reaction and dehydration to form imine cations, followed by 2-azacop rearrangement reaction to form a new imine cation, and then through further hydrolysis and β-elimination to finally obtain L-lysine Acid (Figure 1C). Based on this reaction, the fluorescent protein and bioluminescent protein can be used to detect and image formaldehyde.

The specific preparation method steps of PrAK are as follows (see Figure 2):

的合成。 Compound 2

Synthesis.

According to the literature (Padhi, B.; Reddy, DS; Mohapatra, DK, the gold-catalyzed diastereoselective synthesis of 2,6-trans-disubstituted tetrahydropyran derivatives: synthesis of double chain amides A and B Application of C1-C13 fragments, RSC Advances 2015, 5(117), 96758-96768) to synthesize compound 1. At 0°C, to a solution of compound 1 (17.44 g, 89.8 mmol) in 100 mL THF was added 8.9 mL propylamine (107.7 mmol). The reaction mixture was stirred at 0°C for 30 minutes, then allyl boronic acid pinacol ester (25.3 mL, 134.7 mmol) was added, and the reaction mixture was heated to ambient temperature and stirred for 10 h. The reaction was quenched and then extracted with EA (3×100 mL). The combined organic layer was washed with brine (100 mL), dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/hexane=1:10) to obtain compound 2 (16.66 g, 67% yield). ¹ H NMR (400MHz, CDCl ₃ ) δ 7.20 (d, J = 8.5 Hz, 2H), 6.81 (d, J = 8.6 Hz, 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 = 7.4 Hz, 3H). ¹³ C NMR ( _{101 MHz, CDCl 3} ) δ 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: C ₁₇ H ₂₈ NO ₂ [M+H] ⁺ 278.2120 (calculated value), 278.2115 (measured value).

的合成。 Compound 3

Synthesis.

To a solution of compound 2 (16.66 g, 60 mmol) in 100 mL THF was added Boc anhydride (20.7 mL, 90 mmol), and the reaction was stirred at 25° C. for 8 h. Then TLC (10% EtOAc/hexane) showed that compound 2 had Completely consumed. The reaction mixture was _{diluted with saturated Na 2} CO ₃ and extracted with EtOAc. The combined organic layer was _{dried over Na 2} SO ₄ , filtered, and concentrated under reduced pressure to obtain the Boc-protected crude product as a colorless oil, which was redissolved in CH ₂ Cl ₂ (100 mL) and used directly for the next step. step. At 0°C, water (10 mL) and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ; 34 g, 150 mmol) were added to the solution in portions. The reaction mixture was stirred at 25°C for 2 h, quenched with saturated aqueous NaHCO ₃ (200 mL), and diluted with water (100 mL) and CH ₂ Cl ₂ (200 mL). The resulting mixture was stirred vigorously for 2 hours. The layers were separated, and the aqueous layer was extracted with CH ₂ Cl ₂ (3×150 mL). The combined organic layer was washed with brine (200 mL), dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/hexane=2:3) to obtain alcohol 3 as a colorless oil (13 g, 84% yield in two steps). ¹ H NMR (300MHz, CDCl ₃ ) δ5.74 (ddt, J = 17.0, 10.2, 6.7 Hz, 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). ¹³ C NMR ( _{101 MHz, CDCl 3} ) δ 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: C ₁₄ H ₂₇ NO ₃ Na[M+Na] ⁺ 280.1889 (calculated value), 280.1883 (measured value).

的合成。 Compound 4

Synthesis.

_{At 0°C, to a CH 2} Cl ₂ (30 mL) solution of compound 3 (3.22 g, 12.5 mmol) was added pyridine (1.35 mL, 16.8 mmol) and 4-nitrophenyl chloroformate (3.4 g, 16.8 mmol) . The reaction mixture was stirred at 25°C for 10 h and quenched with water. The layers were separated, and the aqueous layer was extracted with CH ₂ Cl ₂ (50 mL). The combined organic layer was washed with saturated _{aqueous NH 4} Cl (3×30 mL) and brine (50 mL), dried over anhydrous Na ₂ SO ₄ , and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/hexane=20:1) to obtain compound 4 (3 g, 57% yield) as a colorless oil. ¹ H NMR (300MHz, CDCl ₃ ) δ8.25 (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 = 7.4 Hz, 3H). ¹³ C NMR (75MHz, CDCl ₃ ) δ 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: C ₂₁ H ₃₀ N ₂ O ₇ Na[M+Na] ⁺ 445.1951 (calculated value) 445.1946 (measured value).

的合成。 Compound 5

Synthesis.

At 0°C, to a solution of Na-Boc-L-Lys (348mg, 1.41mmol) in DMF (5mL) was added DIPEA (0.47mL, 0.61mmol), DMAP (23mg, 0.19mmol) and compound 4 (396mg, 0.94) mmol) in DMF (5 mL). The reaction mixture was stirred at 25°C for 10 h and quenched with water. The layers were separated, and the aqueous layer was extracted with CH ₂ Cl ₂ (50 mL). The combined organic layer was washed with 1N HCl (3×15 mL) and brine (20 mL), dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (5% MeOH/CH ₂ Cl ₂ ) to obtain white solid compound 5 (317 mg, 64% yield). ¹ H NMR (400MHz, CD ₃ OD) δ 5.75 (td, J = 17.0, 7.0 Hz, 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=7.4 Hz, 3H). ¹³ C NMR (75MHz, CD ₃ OD) δ174.97,162.45,157.70,156.84,156.29,135.41,118.28,116.32,79.90,79.41,79.17,78.26,61.68,53.54,40.16,38.01,37.46,31.21,29.24,27.58, 22.89, 10.71. HRMS: C ₂₆ H ₄₈ N ₃ O ₈ [M+H] ⁺ 530.3441 (calculated value), 530.3467 (measured value).

的合成。 PrAK

Synthesis.

To the solution of compound 5 (317 mg, 0.6 mmol) was added 8 mL of HCl in 1,4-dioxane (4M). The reaction mixture was stirred at 25°C for 2 h. The solvent was removed under reduced pressure to obtain PrAK as a white solid (185.3 mg, 94% yield). ¹ H NMR (400MHz, CD ₃ OD) δ 5.84 (td, J = 16.9, 7.1 Hz, 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). ¹³ C NMR (75MHz, CD ₃ OD) δ 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: C ₁₆ H ₃₂ N ₃ O ₄ [M+H] ⁺ 330.2393 (calculated value), 330.2381 (measured value).

Example 2 In vitro Fmoc-PrAK and formaldehyde reactivity experiment

1) To prepare Fmoc-PrAK, the specific method steps are as follows (see Figure 3):

的合成。 Compound 6

Synthesis.

To a solution of Na-Fmoc-L-Lys (190 mg, 0.474 mmol) in DMF (5 mL) was added DIPEA (100 μL, 0.61 mmol) and compound 4 (143 mg, 0.34 mmol) in DMF (5 mL). The reaction mixture was stirred at 25°C for 10 h and quenched with water. The layers were separated, and the aqueous layer was extracted with CH ₂ Cl ₂ (50 mL). The combined organic layer was washed with 1N HCl (3×15 mL) and brine (20 mL), dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (5% MeOH/CH ₂ Cl ₂ ) to obtain white solid compound 6 (86 mg, 30% yield). ¹ H NMR (300MHz, d ⁶ -DMSO) δ 7.89 (d, J = 7.3 Hz, 2H), 7.73 (d, J = 7.1 Hz, 2H), 7.60 (d, J = 7.8 Hz, 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=6.7 Hz, 3H). ¹³ C NMR (75MHz, d ⁶ -DMSO) δ174.61,156.73,155.43,144.43,144.38,141.30,136.28,128.21,127.64,125.87,120.69,117.32,79.00,78.71,66.17,61.63,54.42,47.25,38.19,37.61 ,32.82,32.31,31.05,29.59,28.62,23.49,22.50,11.99. HRMS: C ₃₆ H ₄₉ N ₃ O ₈ Na[M+Na] ⁺ 674.3417 (calculated value), 674.3412 (measured value).

Synthesis of Fmoc-PrAK.

To the solution of compound 6 (36 mg, 0.055 mmol) was added 3 mL of HCl in 1,4-dioxane (4M). The reaction mixture was stirred at 25°C for 2 h. The solvent was removed under reduced pressure to obtain Fmoc-PrAK (25 mg, 83% yield) as a white solid. ¹ H NMR (500MHz, CD ₃ OD) δ 7.78 (d, J = 7.5 Hz, 2H), 7.66 (t, J = 8.4 Hz, 2H), 7.38 (t, J = 7.4 Hz, 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). ¹³ C NMR (126MHz, CD ₃ OD) δ178.68,178.61,158.68,158.08,145.46,145.27,142.59,132.95,128.75,128.14,126.21,120.90,120.74,67.76,61.51,57.23,56.12,56.06,49.85,48.53, 47.49, 41.50, 35.49, 35.40, 33.51, 31.17, 30.52, 30.45, 23.71,20.82, 11.30. HRMS: C ₃₁ H ₄₂ N ₃ O ₆ [M+H] ⁺ 552.3074 (calculated value), 552.3066 (measured value).

2) The reaction of Fmoc-PrAK with formaldehyde

Add formaldehyde solution (final 1mM) to Fmoc-PrAK (0.5mM) solution (PBS and acetonitrile mixed solvent (v/v=1:1)). Incubate the resulting mixture at 37°C for 1 hour, and then use Shimadzu LCMS-2020 The system directly analyzes the reaction mixture by LC-MS. The UV detector is set to 254nm, and the absorption at 360nm is used as the reference. The Alltima reversed-phase C18 column (4.6×250mm, 5μm) is filled with water (containing 0.1% TFA) and The sample was eluted with a linear gradient of acetonitrile (70% acetonitrile within 15 minutes) at a flow rate of 0.8 mL/min. ESI and APCI ionization sources were used for MS analysis.

The reaction between Fmoc-PrAK and formaldehyde was analyzed by LC-MS, and the results confirmed the production of the product Fmoc-Lys and aldehyde intermediates (Figure 4), thus verifying that PrAK has a 2-azakop rearrangement for formaldehyde Reactivity may be used for formaldehyde detection.

Example 3 Site-specific introduction of unnatural amino acid PrAK into protein

1) PrAKRS sequence optimization and screening

In order to obtain the active site mutant of pyrrolysyl tRNA synthetase capable of inserting PrAK, the present invention modeled PrAK calculation simulation into the pyrrolysine binding pocket of pyrrolysyl tRNA synthetase, and noted that PrAK’s The side chain may conflict in space with many residues around the pocket (for example, Y306, L309, C348, M350, I405, and I413V). Therefore, a series of active site mutants based on wild-type pyrrolysyl tRNA synthetase were constructed by mutating these residues into amino acids with less steric hindrance through rational design, as shown in the table below.

Mutation 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 present invention co-transforms the pLX-EGFP-Y39TAG plasmid and the pBX-PylRS active site mutant plasmid into the E. coli strain BL21. The transformed monoclonal bacteria were cultured in LB medium containing kanamycin (40μg/mL) and chloramphenicol (34μg/mL) at 37℃ overnight with shaking, and then inoculated with kanamycin at a dilution of 1:100. Cultured in fresh TB medium (pH=8.0) containing sodium sulfate (40μg/mL) and chloramphenicol (34μg/mL) and shaking at 37°C. When the OD600 reached 0.6, 2mM PrAK was added to the bacterial culture. After incubating for 1 hour, protein expression was induced by 1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) at 37°C for 10 hours. The fluorescence intensity of a single bacterial culture was measured on a microplate reader and compared with a control culture without PrAK.

Happily, the present invention discovered a pyrrolysyl tRNA synthetase mutant (Y306A, L309A, Y384F, named PrAKRS), whose sequence is shown in SEQ ID NO. 1, which can effectively introduce PrAK EGFP-Y39TAG (Figure 5).

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

2) Incorporate PrAK into E. coli protein

The target plasmid containing the amber stop codon TAG and the pBX-PrAKRS plasmid were co-transformed into E. coli strain BL21(DE3). The transformed bacteria were grown overnight at 37°C in LB medium containing kanamycin (40μg/mL) and chloramphenicol (34μg/mL), and then inoculated with a 1:100 dilution to supplement with kanamycin (40μg /mL) and chloramphenicol (34μg/mL) in fresh TB medium (pH=8.0) and shake culture at 37°C. When the OD600 reached 0.6, 2mM PrAK was added to the bacterial culture. After 1 hour of incubation, protein expression was induced for 10 hours by adding 1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG). Afterwards, the cells were collected by centrifugation and lysed with 4% SDS lysis buffer (4% SDS, 150 mM NaCl, 50 mM triethanolamine, pH 7.4) at 95°C. The resulting cell lysate was centrifuged at 16,000 x g for 5 minutes at room temperature to remove cell debris. The protein concentration was determined by the BCA assay method. Finally, the cell lysates were separated by electrophoresis on a self-made SDS-PAGE gel, and analyzed by immunoblotting and Coomassie brilliant blue staining.

The results show that the full-length EGFP protein will only be expressed when PrAK is present in the medium. Therefore, PrAK successfully integrated into the Y39 and K85 sites of E. coli EGFP (Figures 6A and 6B). In addition, PrAK showed an insertion efficiency comparable to BocK in E. coli (Figure 6A). BocK is N(e)-Boc-L-lysine, which is recognized as an excellent substrate for wild-type MmPylRS.

3) Incorporate PrAK into the protein of mammalian cells

HEK293T cells were seeded on a 12-well plate coated with polylysine and cultured in 1 mL growth medium overnight. On the second day, with or without PrAK (1mM), the target plasmid containing the amber codon TAG (0.65μg per well) and the PylRS plasmid (0.35μg per well) were combined using PEI (2.5μg per well). Transfect into cells. In order to express PrAK-modified protein, pEF1α-FLAG-PrAKRS was used; on the other hand, pEF1α-FLAG-MmPylRS was incorporated into BocK as a control. After 24 hours of transfection, the cells were lysed with 4% SDS lysis buffer containing protease inhibitors and β-mercaptoethanol by sonication and vortexing. The resulting cell lysate was centrifuged at 16,000 x g for 5 minutes at room temperature to remove cell debris. The protein concentration was determined by the BCA assay method. Finally, the cell lysates were separated on a self-made SDS-PAGE gel and analyzed by immunoblotting.

The results show that in mammalian HEK293T cells, the full-length EGFP protein can only be expressed when PrAK is present in the medium, indicating that the gene encoding of PrAK at Y39 and K85 of EGFP has been successfully achieved in mammalian HEK293T cells (Figure 6C).

The above results indicate that PrAK can be specifically encoded into EGFP by the PrAKRS mutant in both E. coli and mammalian cells.

Example 4 Preparation of EGFP-K85PrAK and in vitro formaldehyde detection of EGFP-K85PrAK

1) Preparation of EGFP-K85PrAK

In order to purify EGFP-K85PrAK, the pLX-EGFP-K85TAG-Twin-Strep-tag plasmid and the pBX-PrAKRS plasmid were co-transformed into E. coli BL21 (DE3). The transformed bacteria were grown overnight at 37°C in LB medium containing kanamycin (40μg/mL) and chloramphenicol (34μg/mL), and then inoculated with a 1:100 dilution to supplement with kanamycin (40μg /mL) and chloramphenicol (34μg/mL) in fresh TB medium (pH=8.0), and culture at 37°C. When the OD600 reached 0.6, 2mM PrAK was added to the bacterial culture. After incubating for 1 hour, protein expression was induced by 1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) at 37°C for 10 hours. The cells were collected and lysed with a sonic interference device in buffer W (100mM Tris-HCl, 150mM NaCl, 1mM EDTA, pH 8.0) containing a mixture of protease inhibitors. After centrifugation, the precipitate was dissolved in PBS buffer (PH=8.0) containing 8M urea. Then the EGFP-K85PrAK protein was purified with Strep-Tactin XT Superflow resin and eluted with BXT buffer (W buffer containing 50 mM biotin). Using a linear urea gradient from 6M to 0M, the EGFP-K85PrAK protein was gradually refolded on the column. The SDS-PAGE electrophoresis chart of the purified EGFP-K85PrAK protein is shown in Figure 7.

2) EGFP-K85PrAK in vitro formaldehyde detection

At 37℃, in a 96-well black plate with optical bottom, treat EGFP-K85BocK or EGFP-K85PrAK protein (0.5μM) in 20mM PBS (pH 7.4) with PBS buffer with different concentrations of formaldehyde for 40 minutes. Then add 5mM DTT and 300mM Tris-HCl (pH 7.4), and incubate at 37°C for 2h to quench the excess formaldehyde. Then the fluorescence intensity of each well at 510nm under excitation of 468nm was measured on a microplate reader.

The results show that EGFP-K85PrAK is actually non-fluorescent (Figure 8A), confirming that the K85 position is essential for EGFP fluorescence. In contrast, as the concentration of formaldehyde in the solution increases, there is a clear fluorescence response at the maximum emission peak at 510 nm, which is formaldehyde concentration-dependent (Figure 8A and Figure 9A). At low concentrations of formaldehyde, there is a linear relationship between the fluorescence intensity of EGFP-K85PrAK and the concentration of formaldehyde (Figure 9B). The in vitro detection limit of formaldehyde is estimated to be 25 μM (Figure 9C). Kinetic analysis showed that the fluorescence response of EGFP-K85PrAK to formaldehyde basically reached saturation at about 1 h (Figure 9D).

For selective determination, 0.5 μM EGFP-K85PrAK protein was treated with 2mM formaldehyde or other potentially interfering reactive species (2mM) at 37°C for 40 minutes. Then 5mM DTT and 300mM Tris-HCl (pH 7.4) were added to each sample, and incubated at 37°C for 2h to quench the excess reaction species. Then, the fluorescence intensity at 510nm of each well with three biological replicates under excitation of 468nm was measured on a microplate reader.

The results show that compared to potentially interfering biologically active carbonyl compounds and common oxidizing and reducing molecules in cells (such as H ₂ O ₂ and glutathione), EGFP-K85PrAK has good selectivity to formaldehyde (Figure 8B) .

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

For further verification, the present invention analyzed the protein molecular weight of EGFP-K85PrAK before and after the formaldehyde treatment: the molecular weight (30176.584Da) observed before the formaldehyde treatment is consistent with the theoretical molecular weight of the full-length EGFP-K85PrAK protein without forming a chromophore. The later observed molecular weight (29973.844Da) was consistent with the molecular weight of the wild-type EGFP that had formed a chromophore (Figure 10), confirming that the reaction of PrAK with formaldehyde can reduce the natural lysine residues to form the EGFP chromophore.

Example 5 Detection of formaldehyde in EGFP-K85PrAK live cells

HEK293T cells were seeded on a 12-well plate coated with polylysine and cultured in 1 mL growth medium overnight. On the second day, cells were co-transfected with plasmids pEF1α-FLAG-PrAKRS (0.35 μg per well) and pCMV-EGFP-K85TAG (0.65 μg per well) using PEI (2.5 μg per well). Cells are cultured completely in the presence of PrAK (1 mM). After 24 hours of incubation, the cell culture medium was replaced with fresh Opti-MEM medium without PrAK. After incubating at 37°C and 5% CO ₂ for another 3 hours, add formaldehyde BSS buffer (136.9mM NaCl, 5.37mM KCl, 1.26mM CaCl ₂ , 0.81mM MgSO ₄ , 0.44mM KH ₂ PO ₄ , 0.335mM Na ₂ HPO ₄ , 10 mM PIPES, pH 7.2) washed for 1 hour, and washed 4 times with Opti-MEM medium within 2 hours to remove excess formaldehyde. Then the cells were stained with NucBlue live cell stain, the cells were exchanged to FluoroBrite DMEM, and imaged on a Nikon A1R confocal fluorescence microscope. For the NucBlue channel, a 405nm laser is used as the excitation light, and the fluorescence signal is collected between 425nm and 475nm. For the EGFP channel, a 488nm laser was used as the excitation light, and the fluorescence signal was collected between 500nm and 550nm. In a control experiment, HEK293T cells were co-transfected with plasmids pEF1α-FLAG-Mm-PylRS and pCMV-EGFP-K85TAG in the presence of BocK (0.25mM). For NaHSO ₃ treatment, cells were treated with 1 mM NaHSO ₃ in BSS buffer and incubated with formaldehyde for 1 hour. At least three fields of view per well are randomly selected for each fluorescence imaging experiment. Quantify the fluorescence intensity of each image in Image J and group them for statistical analysis. Use the one-way analysis of variance in GraphPad Prism for statistical analysis of multiple comparisons.

The results show that EGFP-K85PrAK can detect and image formaldehyde with biologically relevant concentrations ranging from 0.1 mM to 1 mM in cells (Figure 11A and Figure 12). In addition, _{pretreatment of cells with NaHSO 3} (formaldehyde scavenger) can significantly reduce the EGFP-K85PrAK fluorescence signal induced by formaldehyde in the cells (Figure 11A).

In order to verify that EGFP-K85PrAK can visualize and image endogenous formaldehyde, the present invention uses tetrahydrofolate (THFA) or 5,10-methylenetetrahydrofolate (5,10-me-THFA) to treat cells expressing EGFP-K85PrAK, THFA and 5,10-me-THFA can be converted to formaldehyde in the folate cycle. In fact, a strong fluorescence signal was indeed observed in the THFA or 5,10-me-THFA treatment group (Figure 11B and Figure 13), indicating that the probe can detect endogenously produced formaldehyde.

In summary, these results indicate that EGFP-K85PrAK is very suitable for fluorescence imaging of physiological levels of formaldehyde in living cells.

Example 6 Preparation of fLuc-K529PrAK and verification of its performance in in vitro detection of formaldehyde

1) Preparation of fLuc-K529PrAK

In order to purify fLuc-K529PrAK, the pLX-fLuc-K529TAG-Twin-Strep-tag plasmid and the pBX-PrAKRS plasmid were co-transformed into E. coli BL21 (DE3). The transformed bacterial cells were grown overnight at 37°C in LB medium containing kanamycin (40μg/mL) and chloramphenicol (34μg/mL), and then inoculated at a dilution of 1:100 to supplemented with kanamycin ( 40μg/mL) and chloramphenicol (34μg/mL) in fresh TB medium (pH=8.0) and cultured at 37°C. When the OD600 reached 0.6, 2mM PrAK was added to the bacterial culture. After incubating for 1 hour, the protein expression was induced by adding 1 mM IPTG at 30°C for 10 hours.

When performing expression analysis experiments, cells were collected and the resulting cell lysate was centrifuged at 16,000 x g for 5 minutes at room temperature to remove cell debris. The protein concentration was determined by the BCA assay method. Finally, the cell lysates were separated on a self-made SDS-PAGE gel and analyzed by immunoblotting and Coomassie brilliant blue staining.

When preparing purified protein, collect the cells in a buffer W (100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0) containing a mixture of protease inhibitors to lyse the bacteria with a sonic interference device. After centrifugation, the supernatant was collected, and then operated according to the instructions. The protein was purified with Strep-Tactin XT Superflow resin and eluted with BXT buffer (W buffer containing 50 mM biotin), and the protein buffer was replaced with PBS buffer by dialysis.

The expression of fLuc-K529PrAK protein in E. coli is shown in Figure 14A. The full-length fLuc protein will only be expressed when PrAK is present in the medium. Therefore, PrAK was successfully integrated into the K529 site of E. coli fLuc. The SDS-PAGE electropherogram of the purified fLuc-K529PrAK protein is shown in Figure 14B. At the same time, the fLuc-K529BocK protein was expressed in the presence of BocK and purified in a similar manner (Figure 14B).

2) fLuc-K529PrAK in vitro detection of formaldehyde

In a 96-well black plate with optical bottom, fLuc-K529BocK or fLuc-K529PrAK protein (0.5μM) was treated with different concentrations of formaldehyde at 37°C for 30 minutes. Then 100mM Tris-HCl (pH 7.4) was added to each well to quench the excess formaldehyde. Use Bright-Lumi Luciferase Assay Kit to measure the luciferase activity on a microplate reader, and perform three biological replicates. Use the chemiluminescence detection mode to capture bioluminescence images on the ChemiDoc imaging system.

The results showed that fLuc-K529PrAK showed a strong dose-dependent bioluminescence response to formaldehyde in an aqueous solution containing ATP and substrate fluorescein (Figure 15A, 15B and Figure 16), while fLuc-K529BocK showed no changes in bioluminescence. (Figures 15A and 15B). At the same time, fLuc-K529PrAK can detect formaldehyde in the physiological concentration range of 0.1-2 mM within 0.5 hours (Figure 15A, 15B and Figure 16).

For selective determination, 0.5μM fLuc-K529PrAK protein was treated in PBS buffer (20mM PBS, pH 7.4) with 0.5mM formaldehyde or other species that might interfere with the reaction at 37°C. Then 100mM Tris-HCl (pH 7.4) was added to each well to quench the excess active reactive species. The luciferase activity was measured in three biological replicates as described above.

The results show that compared to potentially interfering biologically active species and common oxidizing and reducing molecules in cells (such as H ₂ O ₂ and glutathione), fLuc-K529PrAK has good selectivity to formaldehyde (Figure 16D).

Example 7 Detection of formaldehyde in fLuc-K529PrAK live cells

HEK293T cells were seeded on a 96-well black plate with optical bottom coated with polylysine and cultured overnight in 0.2 mL growth medium. On the second day, with or without the addition of 1mM PrAK, the plasmids pEF1α-FLAG-PrAKRS (35ng per well) and pCMV-fLuc-K529TAG (65ng per well) were used in complete cell growth medium with PEI (250ng per well) Co-transfected cells. After 24 hours of incubation, the cell culture medium was replaced with fresh Opti-MEM medium without PrAK. For time-dependent luminescence imaging of formaldehyde, cells were treated with 0.5 mM formaldehyde in BSS buffer for the specified time. For dose-dependent luminescence imaging, cells were treated with different concentrations of formaldehyde in BSS buffer for 1 hour. Use Bright-Lumi Luciferase Assay Kit to measure luciferase activity on a microplate reader. Use the chemiluminescence detection mode to capture bioluminescence images on the ChemiDoc imaging system.

The results showed that in live HEK293T cells expressing fLuc-K529PrAK, the luminescence signal of the formaldehyde treatment group was significantly enhanced, and the formaldehyde was dose-dependent (Figure 15C, 15D and Figure 17). At the same time, in live HEK293T cells expressing fLuc-K529BocK, no increase in luminescence signal was observed in the formaldehyde-treated group (Figures 15C and 15D).

In summary, these data indicate that fLuc-K529PrAK is a genetically encoded bioluminescent probe that can detect physiological levels of formaldehyde in vitro and on living cells.

In summary, the present invention has successfully developed a formaldehyde-reactive lysine analogue PrAK, based on its prepared fluorescent probes and bioluminescent probes encoded by EGFP-K85PrAK and Luc-K529PrAK, which can be used for detection And formaldehyde in imaging solutions and living cells.

The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications according to the present invention, as long as they do not deviate from the spirit of the present invention, they shall fall within the scope of the appended claims of the present invention.

Claims (29)

A class of unnatural amino acids, its general structural formula is as follows:

Wherein R is alkyl; preferably, R is methyl, ethyl, propyl or benzyl. The unnatural amino acid of claim 1, wherein the R is a propyl group, and the unnatural amino acid is PrAK, and its structural formula is as follows:

The unnatural amino acid of claim 2, wherein the unnatural amino acid is a genetically encoded formaldehyde-reactive unnatural amino acid. The preparation method of the unnatural amino acid according to any one of claims 2-3, characterized in that it comprises the following steps: Step 1) The addition reaction of hydroxyl-protected 3-hydroxypropionaldehyde with propylamine and allyl boronic acid pinacol ester to prepare homoallylamine; Step 2) Homoallylamine protects the amine group and removes the hydroxyl protecting group to generate an alcohol compound; Step 3) The alcohol compound is activated by the alcohol hydroxyl group, and is connected with the L-lysine protected by the amine group to form a carbamate compound; Step 4) The carbamate compound removes the amine protecting group to generate an unnatural amino acid. The preparation method of the unnatural amino acid according to claim 4, characterized in that the protection of the hydroxyl group in step 1) is p-methoxybenzyl; preferably, the protection of the amine group in step 2) is tert-butoxycarbonyl (Boc ); More preferably, the activation in step 3) is to use p-nitrophenyl chloroformate to activate the alcoholic hydroxyl group, and the amine protecting group is tert-butoxycarbonyl (Boc) or fluorenylmethyloxycarbonyl (Fmoc). A compound linked to the unnatural amino acid of any one of claims 1-3. A composition comprising the unnatural amino acid of any one of claims 1-3. An expression system, translation system or cell comprising the unnatural amino acid of any one of claims 1-3. A biological macromolecule probe, characterized in that, the biological macromolecule probe is introduced into any one of the unnatural amino acids of claims 1-3 at a specific site of the biological macromolecule; preferably, the biological macromolecule probe The needle is a genetically encoded fluorescent protein probe or a bioluminescent protein probe. The biological macromolecule probe of claim 9, wherein the fluorescent or bioluminescent protein is enhanced green fluorescent protein (EGFP) or firefly luciferase (fLuc), respectively; preferably, the enhanced green fluorescent protein (EGFP) or one of the key lysine sites of firefly luciferase (fLuc) is specifically introduced into 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, that is, the genetically encoded formaldehyde fluorescent probe and bioluminescence probe are EGFP-K85PrAK and fLuc-K529PrAK. A composition comprising the biological macromolecular probe of any one of claims 9-10. An expression system, translation system or cell comprising the biological macromolecular probe of any one of claims 9-10. Application of the biological macromolecule probe of any one of claims 9-10, or the composition of claim 9, or the expression system, translation system or cell of claim 10, in imaging or detecting formaldehyde in samples . The application according to claim 13, wherein the sample is a biological sample; preferably, the biological sample is a living experimental animal, more preferably, a mouse, zebrafish, nematode, or fruit fly. The application of claim 13, wherein the biological sample is a mammalian cell; preferably, the biological sample is a human HEK293T cell. The application of claim 13, wherein the sample is an in vitro or extracellular sample. A method for preparing a biomacromolecule probe, which is characterized in that the unnatural amino acid according to any one of claims 1 to 3 is introduced into the biomacromolecule at a specific point. The preparation method of claim 17, wherein the biomacromolecule probe is a genetically encoded formaldehyde fluorescent probe or a bioluminescent probe, which converts the unnatural amino acid of any one of claims 1-3 to a fluorescent probe. Protein or luciferase biological macromolecules are introduced at designated sites to prepare genetically encoded formaldehyde fluorescent probes or bioluminescent probes. The method of claim 18, which comprises co-expressing the pyrrolysyl-tRNA synthetase gene, homologously associated tRNA and the need to introduce unnatural amino acids in the host cell in the presence of the unnatural amino acid PrAK The site of PrAK is mutated to a fluorescent protein or luciferase gene with amber stop codon TAG. The method of claim 19, further comprising a purification step to obtain genetically encoded formaldehyde fluorescent probes and bioluminescent probes through purification; preferably, the fluorescent protein with PrAK introduced at a specific site is prepared through affinity purification Or luciferase protein; more preferably, through Ni ²⁺ column or

The column performs affinity purification of the tagged protein. 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 also contains pyrrolysine The acyl-tRNA synthetase gene and its homologous associated tRNA, the second expression vector contains the fluorescent protein or luciferase gene whose unnatural amino acid PrAK is to be introduced and whose specific site is mutated to the amber stop codon TAG; preferably, The host cell is E. coli BL21 (DE3). The method of claim 21, wherein the amino acid sequence of the pyrrolysyl-tRNA synthetase is shown in SEQ ID NO. 1; more preferably, the fluorescent protein is enhanced green fluorescent protein (EGFP) through Orthogonal translation system introduces the original 85th lysine site into the unnatural amino acid according to any one of claims 1-3; or the luciferase is firefly luciferase (fLuc) through The cross-translation system specifically introduces the original lysine position at position 529 into the unnatural amino acid described in any one of claims 1-3. The method for imaging or detecting formaldehyde in biological samples with unnatural amino acids according to any one of claims 1-3, characterized in that it comprises the following steps: 1) The unnatural amino acid described in any one of claims 1-3 is introduced into the fluorescent protein or luciferase biomacromolecule in a biological sample to prepare genetically encoded formaldehyde fluorescent probes and bioluminescent probes; or use the right Require any one of the methods described in 17-22 to prepare genetically encoded formaldehyde fluorescent probes and bioluminescent probes; 2) Using genetically encoded formaldehyde fluorescent probes and bioluminescent probes to detect formaldehyde in biological samples. The method for imaging or detecting formaldehyde in biological samples with unnatural amino acids according to claim 23, wherein the biological samples are living experimental animals, preferably mice, zebrafish, nematodes, and fruit flies; or, the biological samples They are mammalian cells, preferably human-derived HEK293T cells. A pyrrolysyl tRNA synthetase, PrAKRS, whose amino acid sequence is shown in SEQ ID NO.1. A gene encoding the pyrrolysyl tRNA synthetase PrAKRS of claim 25. An application of the pyrrolysyl tRNA synthetase PrAKRS of claim 25 in the site-specific introduction of unnatural amino acids into biological macromolecules; preferably, the unnatural amino acid is the one described in any one of claims 1-3 Natural amino acid; more preferably, the biological macromolecule is fluorescent protein or luciferase. An orthogonal translation system, characterized in that the orthogonal translation system is an E. coli cell, comprising the pyrrolysyl tRNA synthetase PrAKRS of claim 25 and the corresponding homologous associated tRNA. A method for producing a protein containing an unnatural amino acid at a selected position in an orthogonal translation system, which is characterized in that it comprises using the pyrrolysyl tRNA synthetase PrAKRS of claim 25 as an aminoacyl-tRNA synthetase to introduce the non-natural amino acid Natural amino acids, or use the orthogonal translation system described in claim 28 to introduce unnatural amino acids.

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