A glucose-based molecular rotor inhibitor of glycogen phosphorylase as a probe of cellular enzymatic function.

Molecular rotors belong to a family of fluorescent compounds characterized as molecular switches, where a fluorescence on/off signal signifies a change in the molecule's microenvironment. Herein, the successful synthesis and detailed study of (E)-2-cyano-3-(p-(dimethylamino)phenyl)-N-(ß-D-glucopyranosyl)acrylamide (RotA), is reported. RotA was found to be a strong inhibitor of rabbit muscle glycogen phosphorylase (RMGPb), that binds at the catalytic site of the enzyme. RotA's interactions with the residues lining the catalytic site of RMGPb were determined by X-ray crystallography. Spectroscopic studies coupled with theoretical calculations proved that RotA is a molecular rotor. When bound in the catalytic channel of RMGPb, it behaved as a light switch, generating a strong fluorescence signal, allowing utilization of RotA as a probe that locates glycogen phosphorylase (GP). RotA, mono-, di- and per-acetylated derivatives, as well as nanoparticles with RotA encapsulated in polyethylene glycol-poly-L-histidine, were used in live cell fluorescence microscopy imaging to test the delivery of RotA through the plasma membrane of HepG2 and A431 cells, with the nanoparticles providing the best results. Once in the intracellular milieu, RotA exhibits remarkable colocalization with GP and significant biological effects, both in cell growth and inhibition of GP.

Synthesis, Kinetic and Conformational Studies of 2-Substituted-5-(ß-d-glucopyranosyl)-pyrimidin-4-ones as Potential Inhibitors of Glycogen Phosphorylase.

Dysregulation of glycogen phosphorylase, an enzyme involved in glucose homeostasis, may lead to a number of pathological states such as type 2 diabetes and cancer, making it an important molecular target for the development of new forms of pharmaceutical intervention. Based on our previous work on the design and synthesis of 4-arylamino-1-(ß-d-glucopyranosyl)pyrimidin-2-ones, which inhibit the activity of glycogen phosphorylase by binding at its catalytic site, we report herein a general synthesis of 2-substituted-5-(ß-d-glucopyranosyl)pyrimidin-4-ones, a related class of metabolically stable, C-glucosyl-based, analogues. The synthetic development consists of a metallated heterocycle, produced from 5-bromo-2-methylthiouracil, in addition to protected d-gluconolactone, followed by organosilane reduction. The methylthio handle allowed derivatization through hydrolysis, ammonolysis and arylamine substitution, and the new compounds were found to be potent (µM) inhibitors of rabbit muscle glycogen phosphorylase. The results were interpreted with the help of density functional theory calculations and conformational analysis and were compared with previous findings.

Multiscale time-resolved fluorescence study of a glycogen phosphorylase inhibitor combined with quantum chemistry calculations.

A fluorescence study of N1-(ß-d-glucopyranosyl)-N4-[2-acridin-9(10H)-onyl]-cytosine (GLAC), the first fluorescent potent inhibitor of glycogen phosphorylase (GP), in neutral aqueous solution, is presented herein. Quantum chemistry (TD-DFT) calculations show the existence of several conformers both in the ground and first excited states. They result from rotations of the acridone and cytosine moieties around an NH bridge which may lead to the formation of non-emitting charge-transfer states. The fingerprints of various conformers have been detected by time-resolved fluorescence spectroscopy (fluorescence upconversion and time-correlated single photon counting) and identified using as criteria their energy, polarization and relative population resulting from computations. Such an analysis should contribute to the design of new GP inhibitors with better fluorescence properties, suitable for imaging applications.