Novel Insight into the Protonation–Deprotonation Equilibria of Tetracycline and Several Derivatives in Aqueous Solution. II. Analysis of the pH-Dependent Fluorescence Spectra by the SVD Technique

Abstract

In a recent publication (M. O. Schmitt and S. Schneider [1], Z. Phys. Chem. 220 (2006) 441), we have shown that the principal component analysis of the sets of absorption spectra recorded during a titration cycle (pH2 ↔ pH11) generally yields more than four significant eigenvectors and thus provides evidence for conformational changes at various pH-levels. In this contribution we report the results of the corresponding fluorescence titration experiments in Stenhagen buffer. Again we find that in most cases the principal component analysis yields more than four significant eigenvectors as expected if tetracyclines behave like a 3-protic acid with fixed conformation or if the equilibrium between the various tautomers/isomers present at each state of protonation is established fast. The consideration of 5 linear independent species proved necessary and sufficient for a good fit of the recorded fluorescence spectra. These contain up to 3 clearly distinguishable components with different intensity ratios and thus provide a first evidence for the presence of different ground-state species rather than parallel photochemical decay pathways of a single excited state. The pK a-values derived from the fluorescence data agree very well with those obtained from the absorption data, and thus confirm that the prerequisites for applying the principal component analysis to fluorescence data are sufficiently good fulfilled if the excitation wavelength is chosen properly. Some changes of conformation, which give rise to only small changes of the absorption spectra, result in larger changes of the emission spectra. This allows conclusions on the mechanisms of excited state intra-molecular proton transfer (ESIPT) in the protonated and neutral species. The fluorescence of the mono- and di-anions, on the other hand, is shown to originate from a geometrically relaxed form without proton transfer. The results reported can explain why titration experiments in unbuffered solution do not produce reproducible results. Furthermore, they show that the spectra recorded at a certain pH are weighted ensemble averages and can not be related to the results of quantum-chemical calculations performed for a specific molecular structure. The reactivity of tetracyclines in alkaline solution varies strongly with the geometry/conformation adopted by the di-anions and therefore depends on “titration history”.