Fluorescent molecular rotors as sensors for the detection of thymidine phosphorylase.

Three new fluorescent molecular rotors were synthesized with the aim of using them as sensors to dose thymidine phosphorylase, one of the target enzymes of 5-fluorouracil, a potent chemotherapic drug largely used in the treatment of many solid tumors, that acts by hindering the metabolism of pyrimidines. 5-Fluorouracil has a very narrowtherapeutic window, in fact, its optimal dosage is strictly related to the level of its target enzymes that vary significantly among patients, and it would be of the utmost importance to have an easy and fast method to detect and quantify them. The three molecular rotors developed as TP sensors differ in the length of the alkylic spacer joining the ligand unit, a thymine moiety, and the fluorescent molecular rotor, a [4-(1-dimethylamino)phenyl]-pyridinium bromide. Their ability to trigger an optical signal upon the interaction with thymidine phosphorylase was investigated by fluorescent measurements.

Synthesis, characterization and inclusion into liposomes of a new cationic pyrenyl amphiphile.

The aggregation properties of a new cationic fluorescent amphiphile tagged on the hydrophobic tail with a pyrene moiety and bearing two hydroxyethyl functionalities on the polar headgroup were investigated by fluorescence experiments as pure components or in mixed liposomes containing an unsaturated phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine, at different molar ratios. The obtained results put in evidence that the conformation and the miscibility of the lipids in the aggregates strongly influence the excimer/monomer ratio. Mixed monolayers at the same composition were investigated by Langmuir compression isotherms to deepen the understanding of lipid organization and miscibility, both in the polar and in the hydrophobic regions. The presence of two hydroxyethyl functionalities on the polar headgroup of the newly synthesized amphiphile exerts a shielding effect of the charge of the amphiphile increasing the compressibility of lipid components in contrast with the disturbing effect of the unsaturated acyl chains of the phospholipid.

Kinetics and mechanistic study of competitive inhibition of thymidine phosphorylase by 5-fluoruracil derivatives.

In a previous investigation, cationic liposomes formulated with new 5-FU derivatives, differing for the length of the polyoxyethylenic spacer that links the N(3) position of 5-FU to an alkyl chain of 12 carbon atoms, showed a higher cytotoxicity compared to free 5-FU, the cytotoxic effect being directly related to the length of the spacer. To better understand the correlation of the spacer length with toxicity, we carried out initial rate studies to determine inhibition, equilibrium and kinetic constants (KI, KM, kcat), and get inside inhibition activity of the 5-FU derivatives and their mechanism of action, a crucial information to design structural variations for improving the anticancer activity. The experimental investigation was supported by docking simulations based on the X-ray structure of thymidine phosphorylase (TP) from Escherichia coli complexed with 3'-azido-2'-fluoro-dideoxyuridin. Theoretical and experimental results showed that all the derivatives exert the same inhibition activity of 5-FU either as monomer and when embedded in lipid bilayer.

Molecular Packing in Langmuir Monolayers Composed of a Phosphatidylcholine and a Pyrene Lipid.

Pyrene lipids are useful tools to investigate membrane organization and intracellular lipid trafficking. The molecular interactions controlling the organization of lipid monolayers composed of a cationic amphiphile tagged with a pyrene residue and a saturated or unsaturated phospholipid, namely, 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphocholine, were investigated by Langmuir trough isotherms to understand how the molecular structure of the components and their relative amount affect the physicochemical properties of lipid monolayers. The obtained results show that the cationic headgroups and unsaturation of hydrophobic chains strongly affect the organization of the lipid monolayer as a function of the amount of components. On the other hand, the presence of the pyrene moiety does not seem to have a marked influence on the interaction within lipid assembly.