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09:44 min
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October 15th, 2019
DOI :
October 15th, 2019
•0:04
Title
0:57
Clickable Caged Amine Preparation
1:53
Functional Unit Installation
3:12
Quantum Efficiency Measurements at 350 nm
3:59
HaloTag Ligand Targeting
6:28
Photomediated Kinase Localization Modulation
7:33
Results: Physical, Photochemical, and Functional Properties of Clickable Caged Compounds
9:05
Conclusion
필기록
The overall goal of this procedure is to provide a practical method for the preparation of photocaged compounds with additional properties such as water solubility or cellular targeting ability. Using a simple synthetic procedure, this technique can be used to expand the laboratory over caged compounds and to make caged compounds with additional properties without compromising their photosensitivity. This synthesis can be performed in a standard laboratory as our clickable caged compounds are stable to photo-irradiation when dissolved in non-nucleophilic organic solvents such as dichloromethane or dimethyl sulfoxide.
Demonstrating the procedure with Hirona Sasaki will be Akinobu Suzuki, a post-doc, Hanami Aoki and Rei Watahiki, grad students from the laboratory. Begin by adding 709.6 milligrams of paBhc methanol and 397.6 milligrams of n n-carbonyldiimidazole into a 30-milliliter round-bottomed flask. Add dry six milliliters of dichloromethane to the flask and stir the solution at ambient temperature for one hour.
At the end of the incubation, add 342.8 milligrams of 4-Dimethylaminopyridine and 552 microliters of tert-butyl six-amino hexyl carbamate, and stir the solution at ambient temperature for an additional three hours. Then use a rotary evaporator under vacuum to remove the solvent and other volatile materials, and use silica gel flash column chromatography to purify the residue directly. To install a functional unit into the clickable cage compound, dissolve 249 milligrams of copper two sulfate pentahydrate in 10 milliliters of ion-exchanged water to give a 0.1 molar copper sulfate solution.
Dissolve eight milligrams of two prime paBhc mock paclitaxel, 7.5 milligrams of tris(3-hydroxypropyltriazolylmethyl)amine, 162.4 milligrams of sodium-l-ascorbate, and 3.1 milligrams of 15-chloro-3, 6, 9-trioxypentyldecyl azide in a mixed solvent of 2.5 milliliters of 0.1 molar phosphate buffer and 0.5 milliliters of dimethyl sulfoxide. Next, add 81.2 microliters of 0.1 molar copper sulfate solution to the reaction mixture and stir the mixture at ambient temperature for 80 minutes, monitoring the progress of the reaction with HPLC. At the end of the reaction, resolve the precipitants with 3.5 milliliters of a 75%acetyl nitryl water solution and apply the resulting solution directly to the semi-preparative HPLC system to purify the desired product.
For quantum efficiency measurements, under fluorescent lamps covered with UV light cut off filter, dilute the sample stock solution in 10 microliters of dimethyl sulfoxide with 10 milliliters of KMOPS buffer. Transfer an aliquot of the solution into the same test tube used in the photo reaction of the chemical actinometer. Irradiate the sample solution with 350 nanometer light for five seconds, removing 50 microliter aliquots from the irradiated solution periodically for analysis by HPLC.
Then determine the irradiation time in seconds in which 90%of the starting material reacted by fitting plots of the time-dependent disappearance of the starting material. For HaloTag ligand targeting of a clickable caged compound, harvest the target cell population of interest with an appropriate cell dissociation reagent and re-suspend the cells in a 2.5 times 10 to the fifth cells per milliliter of DMEM concentration. Then seed approximately 10 to the fifth cells per dish into 35 milliliter glass bottom dishes for an overnight incubation at 37 degrees celsius and 5%carbon dioxide.
The next morning, dilute 14 micrograms of the PC DNA three halo epidermal growth factor receptor plasma DNA in a 1.5 milliliter micro centrifuge tube containing 700 microliters of reduced serum medium. Next, add five microliters of the lipofection reagent in 150 microliters of reduced serum medium to each of four tubes and allow the tubes to stand at ambient temperature for five minutes. At the end of the incubation, add 150 microliters of diluted plasma DNA to each of the diluted lipofection reagent samples and incubate the samples at ambient temperature for an additional five minutes.
At the end of the incubation, rinse the cells with two milliliters of PBS per dish and add 1.5 milliliters of reduced serum medium to each culture. Add 150 microliters of the plasmid lipofection reagent complex to each dish and return the cells to the cell culture incubator for 48 hours. At the end of the incubation, aspirate the supernatants and add one milliliter of freshly prepared DMEM supplemented with two micromolar paBhc hex fitzi halo to each dish.
After 30 minutes at 37 degrees celsius, aspirate the medium containing the caged compound and rinse the cells two times with one milliliter of PBS plus to remove any unbound compounds. Add 500 microliters of reduced serum medium to each dish and return the cells to the cell culture incubator for another 30 minutes to remove the compounds that entered the cells. At the end of the incubation, rinse the cells two times with one milliliter of PBS plus per dish and add one milliliter of medium without phenol red to each culture.
Then record the florescence images by laser scanning confocal florescence microscopy. For photo-mediated modulation of the kinase localization using a clickable caged compound, seed approximately five times 10 to the fifth TOC-A-one cells in two milliliters of Ham's F12 medium per 35 milliliter glass bottom dish. The next day, transfect the cells with the plasmid coding for GFP diacylglycerol kinase gamma for 48 hours as just demonstrated.
After the end of the transfection, replace the transfection supernatant with two milliliters of reduced serum medium. Add 20 microliters of 100 times paBhcAA working solution to the cells for a five to 60 minute incubation at 37 degrees celsius and 5%carbon dioxide. At the end of the incubation, place the dish on the objective stage of an inverted fluorescent microscope equipped with a dual light source fluorescence illuminator.
Image the cells every 10 seconds for 10 minutes while irradiating the cells at the appropriate experimental time points and wave lengths according to the experimental protocol. Using this method as demonstrated, clickable caged compounds of some biologically interesting molecules, including paclitaxel and arachidonic acid, can be successfully synthesized. Additional properties such as the water solubility and cellular targeting ability can be introduced into the paBhc mock paclitaxel via the copper one catalyzed cyclization click reaction.
These clickable caged paclitaxels can then be photolysed to produce their parent compounds upon irradiation at 350 nanometers. The physical and photochemical properties of the clickable caged compounds are summarized in the table. In live cell experiments, the targeting of paBhc hex fitzi halo to cultured mammalian cells transiently expressing a HaloTagged protein and epidermal growth factor receptor induces a green florescence signal from the fluorescein moiety PAHBHC hex fitzi halo on the cell membrane.
Further, the treatment of CHO-K1 cells transiently expressing GFP diacyl glycerol kinase gamma with arachidonic acid causes modulation of the subcellular localization of diacyl glycerol kinase gamma. Similar changes in diacyl glycerol kinase gamma localization are also observed in paBhc-AA treated cells after UV light exposure. When performing the caging experiments in aqueous solutions increasing culture medium, be sure to always work under fluorescent lamp with short wave length capped filter.
Following this procedure, you can study caged compounds that have been abundant for use in biological experiments due to a lack of water solubility, membrane permeability or cellular targeting ability.
A protocol for the synthesis and measurement of the photochemical properties of modular caged compounds with clickable moieties is presented.
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