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Synthesis, biological investigations and molecular docking studies of nonantibiotic and nontetracyclic inducers that feature a minimal key motif of the natural lead tetracycline are presented. The diarylpropane-1,3-dione motif was identified as the minimal substructure responsible for TetR induction by tetracyclines. The first nontetracyclic surrogates of the natural tetracyclines displayed significant inducing effects for TetR(BD)S135L, whereby the chlorohydroxyphenyl-substituted beta-diketone 31 displayed the highest activity. Interestingly, antibiotic activity could not be detected for 31. Homology modeling based on the X-ray structure of 7-chlorotetracycline bound to TetR indicated analogous binding modes for the natural inducer and the synthetic diarylpropane-1,3-dione derivatives.

A combination of structures, energies, and spectral data calculated using density functional theory (DFT) with experimental NMR data has been used to assign conformational equilibria for tetracycline and 5a,6-anhydrotetracycline in water at pH 1, 7, and 10 and in chloroform (5a,6-anhydrotetracycline) and methanol (tetracycline). The results suggest that tetracycline always prefers the extended conformation but that 5a,6-anhydrotetracycline exists in water as a mixture of the two conformers and in chloroform exclusively in the twisted conformation. The conformational equilibria are also shown to be pH dependent.

Semiempirical molecular orbital theory has been used for a systematic scan of the binding positions for a Mg2+ ion with 5a,6-anhydrotetracycline taking both conformational flexibility and possible different tautomeric forms into account. The magnesium ion has been calculated alone and with four or five complexed water molecules in order to simulate the experimental situation more closely. The results are analyzed by comparing the behavior of the title compound with that of tetracycline itself and possible causes for the stronger induction of the Tetracycline Receptor (TetR) by 5a,6-anhydrotetracycline than by tetracycline are considered. Energetically favored 3D-structure of the zwitteranionic 5a,6-anhydrotetracycline magnesium complex in solution.

The binding motif (pharmacophore) for induction and the changes in the structure of the binding site that accompany induction have been determined from molecular-dynamics simulations on the tetracycline-repressor signal-transduction protein. The changes and the induction mechanism are discussed and compared with conclusions drawn from earlier X-ray structures. The differences in inducer strength of tetracycline and 5a,6-anhydrotetracycline are discussed with respect to their interaction in the MD simulations.

The binding of anhydrotetracycline (atc) in wild-type TetR(D), TetR(B), and four single tryptophan mutants of TetR(B) was investigated by UV/vis absorption, steady state, and time-resolved fluorescence spectroscopy. From absorption titration experiments with Mg2+, we conclude that binding of one [atc-Mg]+ complex in the homodimer causes changes in the protein conformation around the second binding pocket. In the presence of absence of Mg2+, several different groups of atc-protein arrangements must exist, each with a characteristic atc fluorescence decay time. Taking into account the results of molecular dynamics (MD) simulations, we propose as one possible origin for such a differentiation teh extent of hydrogen bonding between atc and the surrounding amino acids. Binding of Mg2+ should change the arrangement of the surrounding amino acids such that some of the excited atc molecules do not undergo the relaxation process typical for free atc. The MD simulations also show that the pattern of intra- and intermolecular hydrogen bonding in the two monomeric units is no correlated, thereby leading to different fluorescence kinetics for atc in the two monomeric units. Furthermore, it is suggested that hydrogen bonding between Arg104 and O10 of anhydrotetracycline could regulate the relaxation processes of excited anhydrotetracycline.