In 2004, we used NMR to solve the structure of the minor groove binder thiazotropsin A bound in a 2 : 1 complex to the DNA duplex, d(CGACTAGTCG)2. In this current work, we have combined theory and experiment to confirm the binding thermodynamics of this system. Molecular dynamics simulations that use polarizable or non-polarizable force fields with single and separate trajectory approaches have been used to explore complexation at the molecular level. We have shown that the binding process invokes large conformational changes in both the receptor and ligand, which is reflected by large adaptation energies. This is compensated for by the net binding free energy, which is enthalpy driven and entropically opposed. Such a conformational change upon binding directly impacts on how the process must be simulated in order to yield accurate results. Our MM-PBSA binding calculations from snapshots obtained from MD simulations of the polarizable force field using separate trajectories yield an absolute binding free energy (−15.4 kcal mol⁻¹) very close to that determined by isothermal titration calorimetry (−10.2 kcal mol⁻¹). Analysis of the major energy components reveals that favorable non-bonded van der Waals and electrostatic interactions contribute predominantly to the enthalpy term, whilst the unfavorable entropy appears to be driven by stabilization of the complex and the associated loss of conformational freedom. Our results have led to a deeper understanding of the nature of side-by-side minor groove ligand binding, which has significant implications for structure-based ligand development.

The present work describes the optimization and validation of a highly selective and sensitive analytical method using solid phase extraction and liquid chromatography tandem mass spectrometry (SPE LC-MS/MS) for the determination of some frequently prescribed pharmaceuticals in urban wastewater received and treated by Sharjah sewage treatment plant (STP). The extraction efficiency of different SPE cartridges was tested and the simultaneous extraction of pharmaceuticals was successfully accomplished using hydrophilic-lipophilic-balanced reversed phase Waters^® Oasis HLB cartridge (200 mg/ 6 mL) at pH 3. The analytes were separated on an Aquity BEH C18 column (1.7 µm, 2.1 mm × 150 mm) using gradient elution and mass spectrometric analysis were performed in multiple reactions monitoring (MRM) selecting two precursor ions to produce ion transition for each pharmaceutical using positive electrospray ionization (+ESI) mode. The correlation coefficient values in the linear calibration plot for each target compound exceeded 0.99 and the recovery percentages of the investigated pharmaceuticals were more than 84%. Limit of detection (LOD) varied between 0.1–1.5 ng/L and limit of quantification (LOQ) was 0.3–5 ng/L for all analytes. The precision of the method was calculated as the relative standard deviation (RSD%) of replicate measurements and was found to be in the ranges of 2.2% to 7.7% and 2.2% to 8.6% for inter and intra-day analysis, respectively. All of the obtained validation parameters satisfied the requirements and guidelines of analytical method validation.

DNA-sequence and structure dependence on the formation of minor groove complexes at 5′-XCYRGZ-3′, where Y = T and R = A, by the short lexitropsin thiazotropsin A are explored based on NMR spectroscopy, isothermal titration calorimetry (ITC), circular dichroism (CD) and qualitative molecular modelling. The structure and solution behaviour of the complexes are similar whether X = A, T, C or G and Z = T, A, I (inosine) or C, 5′-CCTAGI-3′ being thermodynamically the most favoured (ΔG = −11.1 ± 0.1 kcal mol⁻¹). Binding site selectivity observed by NMR for 5′-ACTAGT-3′ in the presence of 5′- TCTAGA-3′ when both accessible sequences are concatenated in a 15-mer DNA duplex construct is consistent with thermodynamic parameters (|ΔG|_ACTAGT > |ΔG|_TCTAGA) measured separately for the binding sites and with predictions from modelling studies. Steric bulk in the minor groove for Z = G causes unfavourable ligand–DNA interactions reflected in lower Gibbs free energy of binding (ΔG = −8.5 ± 0.01 kcal mol⁻¹). ITC and CD data establish that thiazotropsin A binds the ODNs with binding constants between 10⁶ and 10⁸ M⁻¹ and reveal that binding is driven enthalpically through hydrogen bond formation and van der Waals interactions. The consequences of these findings are considered with respect to ligand self-association and the energetics responsible for driving DNA recognition by small molecules in the DNA minor groove.