Über hydrophobe Acridinfarbstoffe zur Fluorochromierung von Mitochondrien in lebenden Zellen

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Abstract
10-n-Alkyl-acridinium-orange-chlorides (alkyl-AOs) are excellent dyes for fluorescence staining of mitochondria in living cells. The thermodynamic and spectroscopic properties of the series alkyl=methyl to nonyl have been investigated. The dyes form dimers in aqueous solution. The dimerisation is mainly a consequence of the hydrophobic interaction. The dissociation constant K respectively association constant K −1 of the dimers describes the hydrophobic interaction and therefore the hydrophobic properties of the dye cations. The dissociation constant K=K 0at the standard temperature T=298 K has been determined spectroscopically in aqueous solution. It depends on the length of the alkyl residue n-CmH2m+1 (m=1–9) (Table 2). In addition the standard dissociation enthalpies (energies) Δ H 0 and dissociation entropies Δ S 0 have been determined from the temperature dependence of K (Table 2). With increasing chain length m the thermodynamic parameters K 0, Δ H 0, Δ S 0 decrease. Therefore with growing m the dimers are stabilized. This stabilization is an entropic effect which is diminished by the energetic effect. The change of the thermodynamic parameters with m is in agreement with the concept of hydrophobic interaction and the stabilization of water structure in the surroundings of hydrophobic residues. As one would expect nonyl-AO is the most hydrophobic dye of the series. As an example the spectroscopic properties of nonyl-AO have been determined. We measured the absorption, luminescence and polarization spectra in rigid ethanol at 77 K. Under these conditions alkyl-AOs associate like dyes in Water at room temperature. The spectra depend on the concentration of the solution. In very dilute solution we observe mainly the spectra of the monomers M, in concentrated solution the spectra of the dimers D. The spectra of M and D are characteristically different. The monomers have one long wave length absorption M 1=20.000 cm−1 with resonance fluorescence. In addition there is a long living phosphorescence at 16.600 cm−1. Its polarization is nearly perpendicular to the plane of the AO residue. The dimers have two long wave length absorption bands D 1=18.700 and D 2=21.200 cm−1 with very different intensities. D 1 has very low intensity and is forbitten, D 2 is allowed. D 1 shows fluorescence. Phosphorescence has not been observed. D 1, D2and also M 1 are polarized in the plane of the AO residue. At short wave length absorption and polarization spectra are very similar. From the spectra we constructed the energy level diagram of M and D (Fig. 9). The first excited state of M splits in D in two levels. The level splitting and the transition intensities agree with quantum mechanical model calculations for dimers with parallel or antiparallel molecular orientation. Hydrophobic interaction needs parallel orientation in the dimers of nonyl-AO. In the dimers of AO and of dyes with very short alkyl chains antiparallel orientation may occur.