The Complexation of Tetracycline and Anhydrotetracycline with Mg2+ and Ca2+:  A Spectroscopic Study

Steady-state absorption and emission, circular dichroism (CD), and time-of-flight secondary-ion-mass-spectroscopic (TOF−SIMS) measurements were performed to study the complexation of tetracycline (TC) and anhydrotetracycline (AHTC) with Mg 2+ and Ca 2+ ions, respectively, in aqueous solutions at pH 8.02. The results obtained suggest that Ca 2+ forms a 1:2 ligand:metal complex with TC via chelation through O10−O11 and O12−O1 and induces thereby the extended conformation A of TC, which is stabilized through hydrogen bonding between the deprotonated dimethylamino nitrogen, N4, and OH12a. pH titrations provide evidence that N4 deprotonates in the presence of a 164-fold molar excess of Ca 2+ at approximately pH ⩾ 7.7 ( c TC = 2.1 × 10 -5 M). In contrast to Ca 2+, Mg 2+ binds to N4−O3 and thereby stabilizes the twisted conformation B of TC. TOF−SIMS measurements indicate that a 1:2 ligand:metal complex is formed in addition to the 1:1 complex. The Mg 2+ -induced increase in the fluorescence intensity and the observed changes in the absorption spectra provide evidence that the other Mg 2+ ion binds to the BCD ring system through the deprotonated O11. In contrast to TC, which adopts the twisted conformation B in aqueous solution at pH 8.02, AHTC exhibits the extended conformation A due to slightly lower deprotonation constants. In the presence of Mg 2+, however, the conformational equilibrium is shifted toward the twisted conformation B due to binding of Mg 2+ to N4. TOF−SIMS measurements suggest that a 2:2 ligand:metal complex is formed. AHTC remains in conformation A upon addition of Ca 2+; complexation through O10 can be excluded on the basis of absorption spectroscopic data.