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

A non-natural amino acid, genetic coding technology, applied in the preparation of carbamate derivatives, the preparation of aminohydroxy compounds, the preparation of organic compounds and other directions

Active Publication Date: 2020-10-09
PEKING UNIV SHENZHEN GRADUATE SCHOOL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, protein-based genetically encoded formaldehyde fluorescent probes and bioluminescence probes are still rarely reported.

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] Example 1 Design and preparation of unnatural amino acid lysine analogue PrAK

[0116] 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 ( figure 1 A, 1B). When formaldehyde is not present, the EGFP and fLuc mutants modified by 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. Luminous activity recovery ( figure 1 A, 1B), so as to realize 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.

[0117] Th...

Embodiment 2

[0129] Example 2 In vitro Fmoc-PrAK and formaldehyde reactivity experiment

[0130] 1) Prepare Fmoc-PrAK, the specific method steps are as follows (see image 3 ):

[0131] Compound 6 Synthesis.

[0132] 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. Separate each layer, use CH for water layer 2 Cl 2 (50 mL) extraction. The combined organic layer was washed with 1N HCl (3×15 mL) and brine (20 mL), and washed with anhydrous Na 2 SO 4 Dry and concentrate under reduced pressure. The residue was purified by silica gel column chromatography (5% MeOH / CH 2 Cl 2 ), a white solid compound 6 (86 mg, 30% yield) was obtained. 1 H NMR(300MHz,d 6 -DMSO)δ7.89(d,J=7.3Hz,2H), 7.73(d,J=7.1Hz,2H), 7.60(d,J=7.8Hz,1H), 7.40(dd,J=14.1,6.8 Hz, 2H), 7.33 (dd, J = 14.1, 6.8 Hz, 2H), 7.09 (s, 1H), 5.81-5.57 (m, 1H), 5.05-5.00 (...

Embodiment 3

[0138] Example 3 Site-specific introduction of unnatural amino acid PrAK into protein

[0139] 1) PrAKRS sequence optimization and screening

[0140] In order to obtain the active site mutant of pyrrolysyl tRNA synthetase capable of inserting PrAK, the present invention modeled PrAK computational simulation into the pyrrolysine binding pocket of pyrrolysyl tRNA synthetase, and noted the PrAK 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.

[0141] 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 ...

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Abstract

The 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 formaldehyde detection and imaging. According to the formaldehyde reactive unnatural amino acid PrAK provided by the invention, biomacromolecules such as EGFP (Enhanced Green Fluorescent Protein) and firefly luciferase (fLuc) can be specifically introduced into sites in a protein translation process; a reaction type biomacromolecule formaldehyde fluorescent probe and a bioluminescence probe are constructed, and formaldehyde in a biological sample can be effectively detected or imaged.

Description

Technical field [0001] The invention relates to the field of biological sample detection, in particular to the field of formaldehyde detection in biological samples. [0002] technical background [0003] 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. It is also a common environmental hazard and carcinogen. But what is less known is that formaldehyde can be produced in a series of biological processes in biological systems, including epigenetic regulation, metabolizable energy per carbon unit, and alcohol detoxification. For example, lysine-specific demethylase (LSD) or histone demethylase (JmjC domain-containing histone demethylases, JHDM) can produce formaldehyde by-products by catalyzing the demethylation of histones; one In the metabolism of carbon units, semicarbazide-sensitive amine oxidase (SSAO), which is sensitive to semicarbazide, generates formaldehyde by cataly...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07C271/22C07C269/06C07C269/04C07C271/16C07C213/02C07C217/46C09K11/06C07K14/435C12N9/02C12N9/00C12N15/52C12P21/00
CPCC07C271/22C07C269/06C07C269/04C07C213/02C09K11/06C07K14/43595C12N9/0069C12Y113/12007C12N9/93C12Y601/01006C12P21/00C07C217/46C07C271/16Y02P20/55
Inventor 彭涛张雨晴杜一萌
Owner PEKING UNIV SHENZHEN GRADUATE SCHOOL
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