Glucose-Based Molecular Rotors as Fluorescent Inhibitors and Probes of Glycogen Phosphorylase

Abstract

Herein, the synthesis and photochemical study of (E)-2-cyano-3-(6-(dimethylamino)naphthalen-2-yl)-N-(β-D-glucopyranosyl)acrylamide, a β-D-glucopyranosyl analogue of the widely used molecular rotor julolidine. The new compound is a fluorescent inhibitor of rabbit muscle glycogen phosphorylase with properties of a molecular rotor. Fluorescence measurements in solutions of increasing viscosity determined that the fluorescence intensity increases with the viscosity of the medium, indicating that the new compound exhibits molecular rotor characteristics. Although the compound fluoresces negligibly in aqueous buffer solution, in the presence of increasing amounts of rabbit muscle glycogen phosphorylase, we observed an increase of fluorescence intensity, attributed to the formation of and inhibitor:enzyme complex. Invitro cellular studies were also undertaken, yielding promising preliminary results for the use of the new compound as a fluorescent probe.

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

“…A comparison between RotA ( 2a ) and our previously reported molecular rotor, compound 1 (Fig. 1), 4 reveals several interesting aspects regarding RotA 's properties. Firstly, RotA exhibited a 5-fold increase in fluorescence intensity for a 30-fold increase in viscosity, whereas compound 1 exhibited the same increase in fluorescence for the double increase (60-fold) in solution viscosity.…”

“…Secondly, when bound in the catalytic site of RMGP b , the fluorescence intensity of 1 increased only 3 times, as opposed to 100 times for RotA , relevant to the intensity of free 1 or RotA , respectively, in solution. In fact, the fluorescence intensity of 1 in the catalytic site of RMGP b was smaller than that in a high viscosity solvent, 4 whereas the behavior of RotA was the opposite (compare Fig. 4 with Fig.…”

“…It can be expected that the more extended conjugation of the naphthyl group impedes rotation towards a dark TICT state similar to the M3 conformation in RotA . In fact, the molecular rotor 1 exhibits significant fluorescence even in less viscous solvents, 4 whereas RotA does not. Secondly, when bound in the catalytic site of RMGP b , the fluorescence intensity of 1 increased only 3 times, as opposed to 100 times for RotA , relevant to the intensity of free 1 or RotA , respectively, in solution.…”

“…They usually consist of an electron donating and an electron accepting group linked through a π-conjugate system. 4 They exhibit high fluorescence quantum yield only when free rotation is hindered due to the viscosity or restraints in the molecule's vicinity. When rotation is free, the MRs form twisted intramolecular charge transfer (TICT) states and may follow a non-radiative decay path.…”

“…18 Our work has focused, for almost twenty years, on the synthesis of GP inhibitors, with emphasis on glucose-based analogues that target the catalytic site of the enzyme using rabbit muscle glycogen phosphorylase as a molecular target (RMGPb). 12,[19][20][21][22][23] We recently reported in a preliminary study, 4 that (E)-2-cyano-3-(6-(dimethylamino)naphthalen-2-yl)-N-(β-Dglucopyranosyl) acrylamide (1, Fig. 1) is a fluorescent molecular rotor that interacts with RMGPb with properties of an MR, exhibiting a modest increase of fluorescence when bound to RMGPb.…”