In the treatment of Type II (noninsulin-dependent) diabetes, management of blood glucose levels is critical. One strategy is to delay digestion of ingested carbohydrates, thereby lowering postprandial blood glucose concentration [1]. This can be achieved by inhibiting the activity of pancreatic a-amylase, which mediates the hydrolysis of complex starches to oligosaccharides, and ⁄ or membrane-bound intestinal a-glucosidases, which hydrolyze these oligosaccharides to glucose in the small intestine [1]. Carbohydrate analogues, such as acarbose (1) and miglitol (2) (Fig. 1 Inhibitors targeting pancreatic a-amylase and intestinal a-glucosidases delay glucose production following digestion and are currently used in the treatment of Type II diabetes. Maltase-glucoamylase (MGA), a family 31 glycoside hydrolase, is an a-glucosidase anchored in the membrane of small intestinal epithelial cells responsible for the final step of mammalian starch digestion leading to the release of glucose. This paper reports the production and purification of active human recombinant MGA amino terminal catalytic domain (MGAnt) from two different eukaryotic cell culture systems. MGAnt overexpressed in Drosophila cells was of quality and quantity suitable for kinetic and inhibition studies as well as future structural studies. Inhibition of MGAnt was tested with a group of prospective a-glucosidase inhibitors modeled after salacinol, a naturally occurring a-glucosidase inhibitor, and acarbose, a currently prescribed antidiabetic agent. Four synthetic inhibitors that bind and inhibit MGAnt activity better than acarbose, and at comparable levels to salacinol, were found. The inhibitors are derivatives of salacinol that contain either a selenium atom in place of sulfur in the five-membered ring, or a longer polyhydroxylated, sulfated chain than salacinol. Six-membered ring derivatives of salacinol and compounds modeled after miglitol were much less effective as MGAnt inhibitors. These results provide information on the inhibitory profile of MGAnt that will guide the development of new compounds having antidiabetic activity.Abbreviations HPA, human pancreatic a-amylase; MGA, maltase glucoamylase; MGAnt, maltase glucoamylase N-terminal catalytic domain; pNP, paranitrophenyl; SIM, sucrase isomaltase.

The syntheses of N-alkylated deoxynojirimycin and 1,5-dideoxy-1,5-iminoxylitol derivatives having either a D- or an L-erythritol-3-sulfate functionalized N-substituent are reported. The alkylating agent used was a cyclic sulfate derivative, whereby selective attack of the nitrogen atom at the least hindered primary center afforded the desired ammonium salt. In aqueous solution, these salts were configurationally labile at the ammonium center. Sulfonium and/or selenonium analogues of the ammonium salts were prepared by analogous reactions. The chalcogen salts were obtained as mixtures of diastereomers, separable in some cases, differing only in the stereochemistry at the configurationally stable sulfur or selenium atoms. Proof of configuration and conformation of each compound was obtained by detailed NMR experiments. The compounds are six-membered ring analogues of salacinol, a known sulfonium-salt glucosidase inhibitor. Evaluation of the target compounds for enzyme inhibition of the glucosidase enzyme glucoamylase G2 indicated that these compounds were either inactive or, at best, only weak inhibitors of maltose hydrolysis.

The mycobacterial cell wall is a complex architecture, which has, as its major structural component, a lipidated polysaccharide covalently bound to peptidoglycan. This structure, termed the mycolyl-arabinogalactan-peptidoglycan complex, possesses a core galactan moiety composed of approximately 30 galactofuranosyl (Galf) resides attached via alternating beta-(1-->6) and beta-(1-->5) linkages. Recent studies have shown that the entire galactan is synthesized by the action of only two bifunctional galactofuranosyltransferases, GlfT1 and GlfT2. We report here saturation-transfer difference (STD) NMR spectroscopy studies with GlfT2 using two trisaccharide acceptor substrates, beta-D-Galf-(1-->6)-beta-D-Galf-(1-->5)-beta-D-Galf-O(CH(2))(7)CH(3) (2) and beta-D-Galf-(1-->5)-beta-D-Galf-(1-->6)-beta-D-Galf-O(CH(2))(7)CH(3) (3), as well as the donor substrate for the enzyme, UDP-Galf. Competition STD-NMR titration experiments and saturation transfer double difference (STDD) experiments with 2 and 3 were undertaken to explore the bifunctionality of this enzyme, in particular to answer whether one or two active sites are responsible for the formation of both beta-(1-->5)- and beta-(1-->6)-Galf linkages. It was demonstrated that 2 and 3 bind competitively at the same site; this suggests that GlfT2 has one active site pocket capable of catalyzing both beta-(1-->5) and beta-(1-->6) galactofuranosyl transfer reactions. The addition of UDP-Galf to GlfT2 in the presence of either 2 or 3 generated a tetrasaccharide product; this indicates that the enzyme was catalytically active under the conditions at which the STD-NMR experiments were carried out.

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