Direct Visualization of Glucose Phosphorylation with a Cationic Polythiophene

Abstract

a-D-Glucose is the chief energy source in organisms and plays an important role in the metabolism of most mammalian tissues and cells. Glycolysis, the major route of glucose metabolism, is initiated by the catalytic phosphorylation to D-glucose-6-phosphate (G-6-P) by hexokinase (HK) in the presence of adenosine triphosphate (ATP) and Mg. Phosphorylation of glucose is important for both energy metabolism and biosynthesis in the cell. It accelerates glucose metabolism and prevents glucose from effusing from cells. Furthermore, the rates of ATP-dependent glucose phosphorylation can be used to identify new members of the hexokinases. Glucose phosphorylation is currently measured by coupling to the reduction of NADP+ using glucose-6-phosphate dehydrogenase as an auxiliary enzyme. The generated NADPH is then monitored by absorption spectroscopy and/or HPLC techniques. These methods share the drawback of including a coupled additional enzymatic reaction and are timeconsuming discontinuous assays. As a way to circumvent these limitations, a synthetic multifunctional pore, octakis(Gla-LeuArg-Trp-His-Val-NH2)-p-octiphenylene (SMP), has been used for developing fluorometric assays. Such methods require intricate design and the synthesis of SMPs. These considerations provide motivation for developing simpler and more convenient methods to detect and monitor glucose phosphorylation. Conjugated polymers have been used as the optical platform for chemical and biological detection. Water-soluble polythiophene derivatives display changes in their conformation and aggregation states upon forming interpolyelectrolyte complexes with DNA. Recently, a cationic conjugated polythiophene was used to successfully respond to the presence of ATP, via a combination of electrostatic and hydrophobic interactions, and to detect DNA cleavage. As demonstrated here, it is possible to use these properties for designing a simple, homogeneous, continuous and real-time protocol to assay glucose phosphorylation. The overall strategy is illustrated in Scheme 1. Cationic poly[3-(3′-N,N,N-trimethylamino-1′-propyloxy)-4-methyl-2,5thiophene hydrochloride] (CPT) was chosen as the optical probe. Upon mixing, electrostatic interactions bring together