Monoazides

The unusual reaction of cyanoacetic acid esters with s-triazine monoazides afforded derivatives of a novel coupled heterocyclic system 2-[l,3,5]triazin-2-yl-l,2-dihydro[l,2,3,4]tetrazine-5-carboxylic acid esters <06CHE965>. 

Table 1 shows the gel fractions of 1,2-polybutadiene film containing mono or diazido compound which were irradiated by ultraviolet radiation. The results show that the gel fractions for diazides are 0.77 - 0.82, and 1,2-polybutadiene was crosslinked by dinitrene which was formed by the photodecomposition of diazide. The gel fractions for monoazides have lower values than those for diazides. This means that the crosslinking with monoazides is less effective than that with diazides. 

Although mononitrenes do not act as crosslinking agents themselves, they can generate unpaired electrons in the polymer chains by the abstraction of hydrogen molecules from the polymer. As the result, the polymer chains are crosslinked by the recombinations of these unpaired electrons. Crosslinking by mononitrene in such a way, depends on the activity of the nitrene and may be not necessarily less efficient in some combination of monoazide with the polymer than that by dinitrene as is seen in the case for... 

Iwayanagi (40) recently extended this system into the mid-UV range by changing the sensitizer to an aromatic monoazide compound (4-azidochalcone). Insolubilization of this mid-UV resist does not result in a cross-linked matrix as occurs with the bisazide sensitized MRS. The primary reaction appears to be insertion of the reactive nitrene into a C-H bond on the ring, forming a secondary amine. 

Carbon disulfide and derivs 2 C60-C61 carbon disulfide monoazide 2 C61 see Azidodithiocarbonic acid 1 A632... 

Ethylmalonic acid and derivs 6 E307 ethylmalonic acid monoazide 6 E307... 

Propidium iodide and ethidium monoazide are suspected mutagens and should be handled accordingly. Sodium azide and fixatives are toxic, and should be handled with particular care. 

It should, however, be remembered that fixed cells cannot be stained with propidium iodide for live/dead discrimination. Fixed cells are, in fact, all dead and will therefore all take up propidium iodide even if some were alive and some dead before fixation. Ethi-dium monoazide offers an alternative to propidium iodide if cells will be fixed before flow analysis. It is a dye that, like propidium iodide, only enters dead cells. It has, however, the added advantage of forming permanent cross-links with DNA when photoactivated. Therefore, cells can be stained for surface proteins, incubated with ethidium monoazide under a desk lamp, washed, and then fixed. At the time of acquisition of data on the flow cytometer, red fluorescence will mark the cells that were dead before fixation. 

Other vital stains take advantage of different cellular properties which can be correlated with cellular physiology Propidium Iodide, Ethidium Bromide, Ethidium Monoazide, Calcofluor White have been widely used to indicate the presence of dead eukaryotes or prokaryotes cells. 2-(p-iodophenyl-)3)(p-nitro-phenyl)-5-phenyl tetrazolium chloride (INT) belongs to a class of stains which can be used to determine if a cell or hyphal compartments [180] can maintain an internal reducing environment (Fig. 20a). There are, however, still a large debate about the reliability of those techniques, depending upon the cells under consideration [181]. Calcofluor (Aex = 380 nm, Aem 420 nm) is a specific cell wall stain which enables to counts buds scars on Saccharomyces cerevisiae [29] to estimate the age of a cell. 

Fig. 3. FACScan analysis of C8166 cells. C8166 cells infected with HIV-1, 4 d postinfection, were stained with (A) control FITC-conjugated isotype antibody, (B) ethidium monoazide bromide (EMA), (C) HIV-specific anti-p24 FITC-con-jugated MAb, and (D) dual stained with EMA and anti-p24-FITC. Dual staining allows determination of the proportion of cells that are either live or dead or infected or uninfected and hence the proportion of cells that are both dead and infected, dead and uninfected, live and infected, or live and uninfected. Comparison of the proportion of cells that are dead/live/infected/uninfected with potential antiviral drug-treated and untreated cells will give an indication of whether the drug is selectively toxic for virus-infected cells or cytotoxic regardless of infection. Data were acquired on a Becton Dickinson FACScan and analyzed using WinMDI software.

Addition of dimethyl acetylenedicarboxylate to give pyrazole-3,4,S-tricarboxylic acid dimethyl ester monoazide ... 

Fluorescently labeled plasmid can be quite useful for cellular uptake and distribution studies (112). The main concern with fluorescently tagged DNA is that the presence of the dye molecules may interfere with the interactions between the DNA and the carrier, or the DNA and cellular components. Of equal importance, the binding of the dye to the DNA must be irreversible, so that the dye molecules do not dissociate from the DNA once internalized by the cell. A number of fluorescent DNA-intercalating dyes are available for DNA labeling, such as ethidium bromide, ethidium monoazide, the TO-PRO series, various other cyanine dyes, and many others (82). For example, the membrane-impermeable compound YO-YO... 

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