Bacterial resistance to existing antibiotics poses a serious threat to human health. Because the peptidoglycan layer surrounding bacterial cells is essential for survival, the enzymes involved in peptidoglycan biosynthesis are attractive targets for the design of new antibiotics. Unfortunately, many of these enzymes are difficult to study because substrates to monitor enzymatic activity are either not available or not soluble under suitable assay conditions. These problems can be solved by utilizing synthetic alternative substrates. We recently reported the synthesis of a soluble substrate analogue for MurG, the enzyme that forms the β-(1,4)-N-acetylglucosaminyl-N-acetylmuramyl pentapeptide subunit of peptidoglycan. Using this substrate analogue, we have been able to develop a direct assay to monitor the activity of the enzyme. We now report the purification of Escherichia coli MurG and information on its kinetic properties and substrate requirements in the absence of membranes. This work lays the foundation for detailed mechanistic and structural investigations of this essential bacterial enzyme.

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Ramoplanin (Figure 1) is a cyclic glycolipodepsipeptide antibiotic that kills gram positive bacteria by inhibiting cell wall biosynthesis. Ramoplanin was shown to block the conversion of Lipid I to Lipid II, 1 a reaction that is catalyzed by the intracellular GlcNAc transferase, MurG (Scheme 1). It was proposed that ramoplanin inhibits MurG by complexing Lipid I, which prevents it from being utilized as a substrate. Below we show that ramoplanin also inhibits the polymerization of Lipid II; therefore, we propose that another mechanism by which ramoplanin can kill bacterial cells is through inhibition of the transglycosylation step of peptidoglycan synthesis. Using a synthetic analogue of Lipid II, we present evidence that enzyme inhibition by ramoplanin involves substrate binding. Ramoplanin undergoes a conformational change upon substrate binding, and the resulting complexes self-associate to form fibrils. The significance of fibril formation is discussed.The mechanism of action of ramoplanin has been investigated in permeabilized bacterial cells and membrane preparations by following the incorporation of radiolabel from a precursor into various intermediates along the pathway to peptidoglycan. 1-3 A limitation of these assays is that if one enzymatic step is blocked, then no information can be obtained about subsequent steps. Thus, because ramoplanin prevents the formation of Lipid II, it is not possible to determine whether it also inhibits the polymerization of Lipid II. We reinvestigated the ability of ramoplanin to block Lipid II polymerization using a modified membrane assay 4 in which the transglycosylases are selectively inhibited to permit the buildup of radiolabeled Lipid II. Following removal of the inhibitor, peptidoglycan synthesis commences. The effect of ramoplanin on Lipid II polymerization was evaluated by monitoring the amount of radioactive peptidoglycan formed in the presence of increasing concentrations of ramoplanin. Ramoplanin blocks the polymerization of Lipid II and thus is an inhibitor of the transglycosylation step of peptidoglycan synthesis (Figure 2).Ramoplanin was proposed to act by complexing substrates required for peptidoglycan synthesis. 1 Unfortunately, difficulties in isolating Lipid intermediates from bacterial cells have hindered studies of their interactions with ramoplanin. 5,6 Moreover, the natural Lipid intermediates contain a 55 carbon polyprenol chain that renders them insoluble in water, and thus difficult to use in biophysical studies of complex formation. We recently developed a synthetic route to a soluble Lipid I analogue (1) to use in studying MurG, 7 the GlcNAc transferase that converts Lipid I to Lipid II. Using purified MurG, we have now made the corresponding Lipid II analogue 2 from 1, as shown (Scheme 2). 8,9 Compound 2 is identical to natural Lipid II except that the 55 carbon chain has been replaced with a 10 carbon unit so that the compound is freely water soluble. 10 The ability of ramoplanin to interact with 2 was investigated by NMR (Figure 3). Ti...

MurG, the last enzyme involved in the intracellular phase of peptidoglycan synthesis, is a membrane-associated glycosyltransferase that couples N-acetyl glucosamine to the C4 hydroxyl of a lipid-linked N-acetyl muramic acid derivative (lipid I) to form the beta-linked disaccharide (lipid II) that is the minimal subunit of peptidoglycan. Lipid I is anchored to the bacterial membrane by a 55 carbon undecaprenyl chain. Because this long lipid chain impedes kinetic analysis of MurG, we have been investigating alternative substrates containing shortened lipid chains. We now describe the intrinsic lipid preferences of MurG and show that the optimal substrate for MurG in the absence of membranes is not the natural substrate. Thus, while the undecaprenyl carrier lipid may be critical for certain steps in the biosynthetic pathway to peptidoglycan, it is not required-in fact, is not preferred-by MurG. Using synthetic substrate analogues and products containing different length lipid chains, as well as a synthetic dead-end acceptor analogue, we have also shown that MurG follows a compulsory ordered Bi Bi mechanism in which the donor sugar binds first. This information should facilitate obtaining crystals of MurG with substrates bound, an important goal because MurG belongs to a major superfamily of NDP-glycosyltransferases for which no structures containing intact substrates have yet been solved.

A highly stereoselective palladium-catalyzed O-glycosylation reaction is described. The reaction of a glycal 3-acetate or carbonate with the zinc(II) alkoxide of acceptors establishes the glycosidic linkage under palladium catalysis to give rise to disaccharides as the product in good yields and with high stereoselectivity. In contrast to the Lewis acid mediated Ferrier procedure, the anomeric stereochemistry of this reaction is controlled by the employed ligand. Whereas the use of a complex of palladium acetate and 2-di(tert-butyl)phosphinobiphenyl as the catalyst results in the exclusive beta-glycoside formation, the same reaction using trimethyl phosphite ligand furnishes an alpha-anomer as the major product. The utility of the 2,3-unsaturation present in the resulting glycoside is demonstrated by the further transformations such as dihydroxylation, hydration, and hydrogenation reactions. Thus, the combination of the glycosylation and subsequent functionalization provides a novel entry to saccharides which are otherwise difficult to prepare. The broad scope of the process, mildness of the reaction conditions, and experimental simplicity should make this method a useful tool in synthetic carbohydrate chemistry.

Kendomycin, also known as (-)-TAN 2162, is a novel polyketide-derived ansamycin isolated from Streptomyces sp., which exhibits potent antagonist and agonist activities at the endothelin and calcitonin receptors, respectively. This bacterial metabolite also possesses a strong antibiotic activity against a range of gram-positive and -negative bacteria and cytostatic effects on the growth of human cancer cell lines. When a novel macroglycosidation reaction is employed as the key step, the first enantioselective total synthesis of kendomycin has been accomplished. A Friedel-Crafts-type ring closure of the acyclic precursor containing tetrahydropyran and benzofuran moieties produces the macrocycle as a single stereoisomer in good yield, thus establishing the aryl C-glycosidic linkage of the ansa core. This reaction requires a phenolic glycosyl acceptor and appears to proceed through a rapid O-glycosidation followed by a slow rearrangement to an aryl C-glycoside. The requisite secomacrocycle is prepared by the Pd(0)-catalyzed B-alkyl Suzuki-Miyaura cross-coupling of two subunits, which in turn can be expeditiously assembled from readily available building blocks in a modular fashion.

Stereoselective Palladium-Catalyzed O-Glycosylation Using Glycals. -A new direct palladium-catalyzed O-glycosylation of glycals is presented. The stereoselectivity at the anomeric stereogenic center is controlled by the palladium ligand and not by the steric or electronic nature of substrates. -(KIM, H.; MEN, H.; LEE*, C.; J. Am.

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