Diels−Alder reactions between 1,4-bis(2,4,5-triphenylcyclopentadienone-3-yl)benzene and
either phenylacetylene (model reaction) or 1,4-diethynylbenzene (polymer formation) were studied. NMR
spectra suggest that the main product in the model reaction is the m,m-isomer (up to 83% yield). X-ray
crystal structure analysis convincingly proved the structure of the above isomer. The polymer-forming
reaction was carried out using different concentrations of the monomeric building block and different
reaction times. As a result, branched polyphenylenes with M
w in the range of 1.2 × 104−12 × 104 g mol-1
were obtained. Both the model compound and the polymers were subjected to intramolecular oxidative
cyclodehydrogenation with copper(II) trifluoromethanesulfonate and aluminum chloride. According to
LD-TOF mass spectrometry, the cyclodehydrogenation of the model compound afforded the planarized
polycyclic aromatic hydrocarbon C66H26. This polycyclic aromatic compound was isolated in 91% yield.
The extended π-conjugation and ordering of cyclodehydrogenated products were demonstrated by Raman
spectroscopy.
We report a new technique to detect enzyme activity inside cells. The method based on Fluorescence Lifetime Imaging (FLIM) technology allows one to follow sensor cleavage by proteolytic enzyme caspase-3. Specifically, we use the FLIM FRET of living cells via the confocal fluorescence microscopy. A specially designed lentivector pLVT with the DNA fragment of TagRFP-23-KFP was applied for transduction of A549 cell lines. Computer simulations are carried out to estimate FRET efficiency and to analyze possible steric restrictions of the reaction between the substrate TagRFP-23-KFP and caspase-3 dimer. Successful use of the fuse protein TagRFP-23-KFP to register the caspase-3 activation based on average life-time measurements is demonstrated. We show that the average life-time distribution is dramatically changed for cells with the modified morphology that is typical for apoptosis. Namely, the short-lived component at 1.8-2.1 ns completely disappears and the long-lived component appears at 2.4-2.6 ns. The latter is a fingerprint of the TagRFP molecule released after cleavage of the TagRFP-23-KFP complex by caspase-3. Analysis of life-time distributions for population of cells allows us to discriminate apoptotic and surviving cells within single frame and to peform statistical analysis of drug efficiency. This system can be adjusted for HTS by using special readers oriented on measurements of fluorescence life-time.
Numerous processes in cells can be traced by using fluorescence resonance energy transfer (FRET) between two fluorescent proteins. The novel FRET pair including the red fluorescent protein TagRFP and kindling fluorescent protein KFP for sensing caspase-3 activity is developed. The lifetime mode of FRET measurements with a nonfluorescent protein KFP as an acceptor is used to minimize crosstalk due to its direct excitation. The red fluorescence is characterized by a better penetrability through the tissues and minimizes the cell autofluorescence signal. The effective transfection and expression of the FRET sensor in eukaryotic cells is shown by FLIM. The induction of apoptosis by camptothecine increases the fluorescence lifetime, which means effective cleavage of the FRET sensor by caspase-3. The instruments for detecting whole-body fluorescent lifetime imaging are described. Experiments on animals show distinct fluorescence lifetimes for the red fluorescent proteins possessing similar spectral properties.
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