A genetically encoded formaldehyde-reactive unnatural amino acid, its preparation method and its application
An unnatural amino acid and genetically encoded technology, which is applied in the preparation of carbamic acid derivatives, aminohydroxy compounds, organic compounds, etc., to achieve easy cell-specific or organ-specific targeting operations, easy introduction into living tissues, and stable mark effect
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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 a non-natural amino acid with formaldehyde reactivity, which is specifically inserted into biomacromolecular fluorescent and bioluminescent proteins, and is used to control their fluorescence and bioluminescence ( figure 1 A, 1B). In the absence of formaldehyde, the unnatural amino acid modified EGFP and fLuc mutants had no fluorescence and bioluminescent activity; in the presence of formaldehyde, the unnatural amino acid reacted with formaldehyde to revert to the key lysine residue, resulting in its fluorescence and bioluminescence. Luminescent activity recovery ( figure 1 A, 1B), so as to realize the detection of formaldehyde. In order to achieve the above purpose, the present invention independently designs a formaldehyde-reactive lysine analog PrAK, which is proved by the detection and imaging of formaldehyde in biolo...
Embodiment 2
[0129] Embodiment 2 Reactivity experiment of Fmoc-PrAK and formaldehyde in vitro
[0130] 1) Prepare Fmoc-PrAK, the specific method steps are as follows (see image 3 ):
[0131] Compound 6 Synthesis.
[0132] To a solution of Nα-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 washed with CH 2 Cl 2 (50 mL) for extraction. The combined organic layers were washed with 1N HCl (3×15 mL) and brine (20 mL), 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 ) to obtain white solid compound 6 (86 mg, 30% yield). 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=14.1,6.8Hz,...
Embodiment 3
[0138] Example 3 Site-directed 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 that can be inserted into PrAK, the present invention models PrAK calculation simulation into the pyrrolysine binding pocket of pyrrolysyl tRNA synthetase, and notices that PrAK The side chains may have steric conflicts with many residues surrounding the pocket (e.g. Y306, L309, C348, M350, I405, and I413V). Therefore, by rationally designing and mutating these residues into amino acids with less steric hindrance, a series of active site mutants based on wild-type pyrrolysyl tRNA synthetase were constructed, as shown in the table below.
[0141] mutant group mutation site group 1 L309A, Y384F group 2 L309A, C348S, Y384F group 3 Y384F group 4 Y306A, Y384F Group 5 Y306G, L309G, Y384F Group 6 L309A, Y348F, Y...
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