Intrinsic Fluorescence of the Active and the Inactive Functional Forms of Human Thymidylate Synthase.

The observables associated with protein intrinsic fluorescence - spectra, time decays, anisotropies - offer opportunities to monitor in real time and non-invasively a protein's functional form and its interchange with other forms with different functions. We employed these observables to sketch the fluorometric profiles of two functional forms of human thymidylate synthase (hTS), a homodimeric enzyme crucial for cell proliferation and thus targeted by anticancer drugs. The protein takes an active and an inactive form. Stabilization of the latter by peptides that, unlike classical hTS inhibitors, bind it at the monomer/monomer interface offers an alternative inhibition mechanism that promises to avoid the onset of drug resistance in anticancer therapy. The fluorescence features depicted herein can be used as tools to identify and quantify each of the two protein forms in solution, thus making it possible to investigate the kinetic and thermodynamic aspects of the active/inactive conformational interchange. Two examples of fluorometrically monitored interconversion kinetics are provided.

Excited-state intramolecular proton transfer in a bioactive flavonoid provides fluorescence observables for recognizing its engagement with target proteins.

A benzothiophene-substituted chromenone with promising activity against Leishmania and Trypanosoma species exhibits peculiar fluorescence properties useful for identifying its complexes with target proteins in the microorganism proteomes. The emission spectra, anisotropy and time profiles of this flavonoid strongly change when moving from the free to the protein-bound forms. The same two types of emission are observed in organic solvents and their mixtures with water, with the relative band intensities depending on the solvent ability to establish hydrogen bonds with the solute. The regular emission prevails in protic solvents, while in aprotic solvents the anomalously red-shifted emission occurs from a zwitterionic tautomeric form, produced in the excited state by proton transfer within the intramolecularly H-bonded form. This interpretation finds support from an experimental and theoretical investigation of the conformational preferences of this compound in the ground and lowest excited state, with a focus on the relative twisting about the chromenone-benzothiophene interconnecting bond. An analysis of the absorption and emission spectra and of the photophysical properties of the two emitting tautomers highlights the relevance of the local microenvironment, particularly of the intra- and intermolecular hydrogen bonds in which this bioactive compound is involved, in determining both its steady-state and time-resolved fluorescence behaviour.

Optical and photophysical properties of anisole- and cyanobenzene-substituted perylene diimides.

One- and two-photon absorption cross-sections and spectra and the photophysical properties of eight perylenetetracarboxy-3,4:9,10-diimide (PDI) derivatives are reported and analyzed. The investigated compounds are characterized by direct binding of the phenyl rings of the substituents to the bay positions of the perylene core. They have been designed to test the effects of differences in the electronic nature - electron donating (anisole) or accepting (cyanobenzene) - and binding topology (cis or trans, meta or para disubstitution or tetrasubstitution) of the bay substituents on the above optical and photophysical observables. (TD)DFT and Hückel MO calculations have provided theoretical information on the ground-state geometries, the MOs and the electronic spectra of several model compounds. For tetrasubstituted and cis disubstituted derivatives, strong steric interactions in the bay area determined the preferred conformations, with perylene cores distorted near the substituted bay(s) and a 42-44° twisting of the substituent rings relative to the core, quite irrespective of the electronic nature of the substituents. On the other hand, in trans-disubstituted PDI steric hindrance in the bay areas was much weaker and similar in the cyanobenzene and the anisole derivatives. So, the large differences found in the conformational preferences were completely attributable to electronic effects. With electron-accepting cyanobenzene, the substituent rings were found normal to the central planar perylene core, thus enabling the assignment of the moderate spectroscopic effects to inductive interactions. The DFT analysis of the PDI trans-disubstituted with electron-donating anisoles gave quite strongly distorted perylene-core geometries and less twisted (59°) substituent rings. The corresponding increased substituent/core conjugative interactions resulted in new CT allowed electronic transitions and an extremely pronounced solvent-polarity dependence of the emission spectra and intensities. All anisole substituted PDI feature a very fast radiationless decay path in polar solvents, likely related to a relaxation to a charge-separated configuration in the lowest excited-state.

Optoelectronic Properties of A-π-D-π-A Thiophene-Based Materials with a Dithienosilole Core: An Experimental and Theoretical Study.

Two A-π-D-π-A thiophene-based small molecules with a central dithienosilole core and dicyanovinyl (DCV) end groups were synthesized. These compounds differ only by the presence of alkyl and alkylsulfanyl chains, respectively, on the thiophene beta positions. Computational data together with the spectroscopic and electrochemical findings (obtained by means of absorption, steady-state/time-resolved emission techniques, and cyclic voltammetry) revealed that both molecules possess low electronic and optical band gaps, broad absorption spectra, and good stability both in p and n-doping states, which make them suitable for optoelectronic applications. In both compounds, the HOMO-LUMO transition involves an intramolecular charge transfer from the electron-donor dithienosilole unit to the two terminal electron-acceptor DCV groups. A marked positive emission solvatochromism was observed for both molecules and was interpreted on the basis of the symmetry breaking in the S1 excited state. The two synthesized compounds were also compared to their shorter precursors and to similar oligothiophenes to understand how the nature of the building block influences the characteristics of the final materials. Furthermore, it was possible to better understand the contribution of the sulfur atom in modulating the optical properties of the small molecules studied.

SiO2-CaO-P2O5 Bioactive Glasses: A Promising Curcuminoids Delivery System.

In this paper, we report the study of the loading and the release of curcuminoids by bioactive glasses (BG) and mesoporous bioactive glasses (MBG). Through a detailed spectroscopic study, it was possible to determine the amount and the type of molecules released in water and in simulated body fluid (SBF). In particular, curcumin and K2T21 show a good ability to be released in di-keto and keto-enolic form, depending from the pH. However, after 24 h, the amount of pristine curcumin release is very low with a consequent increment of degradation products derived by curcuminoids. The presence of -OH groups on curcuminoids is a fundamental pre-requisite in order to obtain a high loading and release in polar solution such as water and SBF. The substrate on which we loaded the drugs does not seem to affect significantly the loading and the release of the drugs. The environment, instead, affects the release: for all the drugs, the release in SBF, buffered at pH of 7.4, is slightly worse than the release in water (basic pH values).

Light-induced degradation of metsulfuron-methyl in water.

Photodegradation of metsulfuron-methyl, a sulfonylurea herbicide, has been investigated in aqueous solution at different pH and excitation wavelengths. The efficiency of the process has been evaluated through quantum yield determinations. The identification of the photoproducts indicates that the major photochemical pathway is initiated by C-S bond dissociation followed by involvement of water to yield the main final products; the behaviour in water is shown to differ markedly from that in an organic environment.

Dimer-monomer equilibrium of human thymidylate synthase monitored by fluorescence resonance energy transfer.

An ad hoc bioconjugation/fluorescence resonance energy transfer (FRET) assay has been designed to spectroscopically monitor the quaternary state of human thymidylate synthase dimeric protein. The approach enables the chemoselective engineering of allosteric residues while preserving the native protein functions through reversible masking of residues within the catalytic site, and is therefore suitable for activity/oligomerization dual assay screenings. It is applied to tag the two subunits of human thymidylate synthase at cysteines 43 and 43' with an excitation energy donor/acceptor pair. The dimer-monomer equilibrium of the enzyme is then characterized through steady-state fluorescence determination of the intersubunit resonance energy transfer efficiency.