Synthesis, structure determination, spectral properties, and energy-linked spectral responses of the extrinsic probe oxonol V in membranes.

The direct synthesis of bis]3-phenyl-5-oxoisoxazol-4-yl]pentamethineoxonol, which is shown to be the fluorescent probe OX-V (formerly MC-V), is described. The emission lifetime (0.9 +/- 0.1 ns) and the spectral properties of this dye in a number of systems are presented as well as the relative polarizations associated with the transition moments of the observable electronic transitions. The structure of OX-V was determined using elemental analysis and infrared and 1H nuclear magnetic resonance (NMR) spectroscopy. The use of the contact shift reagent, Eu(fod)3-d27, greatly facilitated the interpretation of the NMR results. In aqueous media, the anionic form of OX-V is present virtually exclusively due to the low solubility of the neutral species; formation of the latter species occurs when ethanol or methanol solutions of OX-V are acidiied. Both neutral and anionic dye forms can be detected in chloroform-ethanol solvents. The fluorescence intensity from excitation of the neutral species is an order of magnitude weaker than that from excitation of the anionic form and may result from the formation of excited anions due to the loss of a proton by the neutral species in the excited state. Polarization results indicate that the visible absorption of the dye is due to a single electronic transition. OX-V has been employed as a probe primarily in beef heart submitochondrial particles, reconstituted ATPase vesicles,a nd pigeon heart mitochondria. The energy-linked spectral changes of the probe in these preparations are described and mechanisms proposed for the spectral effects.