Novel Insight into the Protonation–Deprotonation Equilibria of Tetracycline, Sancycline and 10-Propoxy-Sancycline in Aqueous Solution. I. Analysis of the pH-Dependent UV/vis Absorption Spectra by the SVD Technique

Summary

The UV/vis absorption spectra recorded during titration experiments (pH2 ↔ pH11, ΔpH ∼ 0.33) were subjected to a principal component analysis. The aim was to determine by means of the program SPECFIT the number of significant components and to reduce the noise in the spectra. The attempt to achieve a global fit of the “noise-reduced” data was in general successful if 5 or more independent species were considered. The necessity to include a larger number of species than the 4 occuring within the simple protonation-deprotonation scheme of a triprotic acid provides strong evidence for conformational heterogeneity at various levels of protonation. Only if this heterogeneity is taken into account, the analysis yields the correct pK_a-values. For tetracycline, slow conformational changes are suggested to occur in the neutral state. Consequently, tetracycline adopts conformation 2 in alkaline solution if the pH is raised slowly up to pH11, whereas if it is dissolved directly in buffer solution at pH2 or pH11 it adopts conformation 1 (like in the crystal). In this conformation, tetracycline degrades rapidly in a methanol–water mixture around pH11 probably by methylation of the 11,12-β-carbonyl group. Sancycline and 10-propoxy-sancycline are stable at any pH, the conformational changes are proposed to occur preferentially in the anionic state. Because of these conformational changes, the pK_a-values derived from forward and backward titration can differ up to 0.4 pH units. In addition, there is evidence that C10-OH does not dissociate at pH less than 11.

In contrast to a widely accepted hypothesis, 4-epi-tetracycline also changes its conformation in the anionic form. This shows that CD spectra are not suited to draw conclusions on molecular geometry if the total net charge changes.