Results:                                2011                        2012                       2013                       2014                       2015                        2016



Determination of the complexing properties (stoichiometry, association constant and molecular geometry) of β-CD with NSAIDs

a) The complexation between the anionic forms of tolfenamic acid (TA) and flufenamic acid (FF) with β-cyclodextrin has been investigated in solution by 1D and 2D 1H NMR spectroscopy. The constructed Job’s plots, based on the induced chemical shifts of the β-CD, TA, and FF protons, confirm the existence of a bimodal binding in both cases. ROESY experiments reveal the correlation between the H3 and H5 protons of the b-CD cavity and both the aromatic ring protons of TA and FF, supporting the bimodal binding process. On the basis of these experimental data we conclude that we have a simultaneous presence of two 1:1 complexes for TA as well as FF, each one involving the inclusion in the b-CD cavity of a different aromatic end. Based on the evaluated microscopic association constants, we believe that the complexation mainly affects the methyl-phenyl or trifluoromethyl-phenyl ring, and to a lesser extent, the benzoic acid moiety.

b) The complexation process between racemic flurbiprofen and β-cyclodextrin in solution wasinvestigated

by 1D and 2D proton NMR spectroscopy. In the presence of β-cyclodextrin, the aromatic protons of flurbiprofen were the most affected, suggesting a strong involvement of the phenyl groups in the inclusion mechanism. The stoichiometry of the complex was determined by the method of continuous variation, using the chemical induced shifts of both host and guest protons. The association constant, Ka of the obtained complex was calculated and found to be 2483.8 M-1. On the other hand, signals belonging to the protons associated with the carboxyl group are split in the presence of β-cyclodextrin indicating enantiomeric differentiation. Rotating frame NOE spectroscopy, (ROESY), was used to ascertain the solution geometry of the host–guest complex. The result suggested that the flurbiprofen molecule fully penetrates the β-cyclodextrin cavity with the carboxyl group protruding from the primary hydroxyl side and the phenyl group close to the secondary rim.

c) The formation of inclusion complexes between the native cyclodextrins (CDs) and the urea herbicide cycluron has been investigated both in solution and in the solid state. Single-crystal X-ray structures of both the uncomplexed guest and the β-CD·cycluron complex were determined while powder X-ray diffraction was used to confirm complexation between g-CD and cycluron in the solid state. Solution-state complexation between the herbicide and a-, b- and g-CD was established using 1H NMR spectroscopy and isothermal titration calorimetry (ITC). From the 1H NMR spectroscopic studies 1:1 complex stoichiometry was indicated in all cases and association constant values (K) were determined as 228, 3254 and 155 for the complexes a-CD·cycluron, b-CD·cycluron and g-CD·cycluron, respectively. Assigning a 1:1 host–guest ratio, the ITC technique produced K values of the same order as those determined using the spectroscopic method. The thermodynamic parameters DH, DS and DG obtained using ITC provide insights into the driving forces involved during complex formation.

αalbumin (HSA), was investigated by fluorescence quenching technique and isothermal titration calorimetry (ITC). A good linearity of albumin fluorescence quenching in the presence of stavudine was determined. Using the most generally valid equation to analyze fluorescence changes upon formation of a 1:1 complex, we obtained for the dissociation constant the value Kd = (11.4 ±0.23)x10-5 18 M. However, due to contradictory results obtained in ITC experiments, we checked the fluorescence quenching data for the inner-filter effect, the main confounding factor in the observed quenching. Based on the UV/vis absorption data we have corrected the observed fluorescence intensities and concluded, in accordance with ITC results, that stavudine is not binding at all to HSA and the observed quenching effect is entirely caused by a failure to correct for the inner-filter effect.





Text Box: Bioligand – macromolecule intermolecular interactions studied by spectroscopic and calorimetric techniques  
Text Box: National  Institute for R&D of Isotopic and Molecular Technologies Cluj-Napoca