Hydration of proteins profoundly affects their functions. We describe a simple and general method for site-specific analysis of protein hydration based on the in vivo incorporation of fluorescent unnatural amino acids and their analysis by steady-state fluorescence spectroscopy. Using this method, we investigate the hydration of functionally important regions of dehalogenases. The experimental results are compared to findings from molecular dynamics simulations.

The structural basis for the interaction of roscovitine and analogues containing 13 different bioisosteric central heterocycles with the enzyme cyclin-dependent kinase 2 (CDK2) is elucidated. Although all the central scaffolds are very similar to the purine core of roscovitine, the experimentally determined IC values of the inhibitors span three orders of magnitude. By using an extensive computational chemistry approach, the affinities of the inhibitors to CDK2 are determined as calculated binding scores of complexes of the inhibitors with the protein. The interactions of the inhibitors with CDK2 are computationally described by using a hybrid quantum mechanics/semi-empirical quantum mechanics method (QM/SQM), which combines the DFT-D method for the QM part and the PM6-D3H4X method for the SQM part. The solvent effect is described by the COSMO implicit solvation model at the SQM level for the whole system. The contributions of the scaffolds and the individual substituents, quantified and evaluated in relation to conformations of optimized protein-inhibitor complexes, are found not to be simply additive. The inhibitory activity of the selected candidates, including two newly prepared compounds, is tested against CDK2. The results of the calculations are in close agreement with the experimental data.

Time-dependent fluorescence shifts (TDFS) of chromophores selectively attached to proteins may give information on the dynamics of the probed protein moieties and their degree of hydration. Previously, we demonstrated that a coumarin dye selectively labeling the tunnel mouth of different haloalkane dehalogenases (HLDs) can distinguish between different widths of tunnel mouth openings. In order to generalize those findings analogous experiments were performed using a different chromophore probing the same region of these enzymes. To this end we synthesized and characterized three new fluorescent probes derived from dimethylaminonaphthalene bearing a linker almost identical to that of the coumarin dye used in our previous study. Labeling efficiencies, acrylamide quenching, fluorescence anisotropies, and TDFS for the examined fluorescent substrates confirm the picture gained from the coumarin studies: the different tunnel mouth opening, predicted by crystal structures, is reflected in the hydration and tunnel mouth dynamics of the investigated HLDs. Comparison of the TDFS reported by the coumarin dye with those obtained with the new dimethylaminonaphthalene dyes shows that the choice of chromophore may strongly influence the recorded TDFS characteristics. The intrinsic design of our labeling strategy and the variation of the linker length ensure that both dyes probe the identical enzyme region; moreover, the covalently fixed position of the chromophore does not allow for a major relocalization within the HLD structures. Our study shows, for the first time, that TDFS may strongly depend on the choice of the chromophore, even though the identical region of a protein is explored.

Inclusion complexation of an antimicrobial quaternary ammonium salt, carbethopendecinium bromide (SBr, Septonex), with five cyclodextrins was investigated potentiometrically using the prepared membrane electrodes selective to the surface-active carbethopendecinium cations (S+). Relatively strong complexation of the S+ cations with native α-cyclodextrin (α-CD), β-cyclodextrin (β-CD), and their random-substituted derivatives, namely hydroxypropyl-α-cyclodextrin (HP-α-CD), methyl-β-cyclodextrin (M-β-CD), and hydroxypropyl-β-cyclodextrin (HP-β-CD), was evidenced in dilute aqueous solution. In the region where the potentiometrically determined concentration of the free S+ cations remained lower than the critical micelle concentration (cmc) of SBr by a factor of ca ten, formation of the 1:1 complexes with the complexation constants K11 ≈ 104-105 was evaluated by two respective methods, based on a modified linear and a non-linear regression. Deviations from the 1:1 complexation were observed when concentration of the free S+ cations approached cmc of SBr more closely and also in solutions with large excess of α-CD. Comparison of K11 values corroborated the inclusion of the n-C14H29 alkyl chain of the carbethopendecinium cation as the mechanism of its complexation with cyclodextrins. The well-soluble native α-CD with good complex-forming capability towards S+ cations may be especially suitable for possible blocking the undesirable residues of carbethopendecinium bromide.

Perkin condensation with subsequent intramolecular lactonisation as one pot syntheses of 2-(2-oxo-2H-chromen-3-yl)acetic acids VIIa-Xa has been studied. The required acids VIIa-Xa were prepared as precursors of recently discovered compounds possessing antineoplastic activities. Syntheses of VIIa-Xa were carried out using para substituted 2-hydroxybenzaldehydes II-VI, succinic acid anhydride, sodium succinate under thermal or microwave conditions. Significant shortening of the reaction time under microwave irradiation was observed (18–50 min instead of 1.5–5 h of heating). Microwave assisted reactions proceeded more smoothly to give higher yield of the required products VIIa-Xa (31–61 %) compared to those under classical thermal conditions e.g. 21.8 % for IXa (Hurenkamp et al., 2007). Seven reaction by-products were isolated and determined as 2H,2′H-3,3′-bichromene-2,2′-diones VIIb-Xb and (E)-3-(2-hydroxystyryl)-2H-chromen-2-ones VIIc-IXc.