Alternate synthesis to d-glycero-ß-d-manno-heptose 1,7-biphosphate.

d-glycero-ß-d-manno-heptose 1,7-biphosphate (HBP) is an enzymatic intermediate in the biosynthesis of the heptose component of lipopolysaccharide (LPS), and was recently revealed to be a pathogen-associated molecular pattern (PAMP) that allows detection of Gram-negative bacteria by the mammalian immune system. Cellular detection of HBP depends upon its stimulation of a cascade that leads to the phosphorylation and assembly of the TRAF-interacting with forkhead-associated domain protein A (TIFA), which activates the transcription factor NF-κB. In this note, an alternate chemical synthesis of HBP is described and its biological activity is established, providing pure material for further assessing and exploiting the biological activity of this compound.

Sialyltransferase inhibitors: consideration of molecular shape and charge/hydrophobic interactions.

In order to evaluate the importance of molecular shape of inhibitor molecules and the charge/H-bond and hydrophobic interactions, we synthesized three types of molecules and tested them against a sialyltransferase. The first type of compounds were designed as substrate mimics in which the phosphate in CMP-Neu5NAc was replaced by a non-hydrolysable, uncharged 1,2,3-triazole moiety. The second type of compound contained a 2-deoxy-2,3-dehydro-acetylneuraminic moiety which was linked to cytidine through its carboxylic acid and amide linkers. In the third type of compound the sialyl phosphate was substituted by an aryl sulfonamide which was then linked to cytidine. Inhibition study of these cytidine conjugates against Campylobacter jejuni sialyltransferase Cst 06 showed that the first type of molecules are competitive inhibitors, whereas the other two could only inhibit the enzyme non-competitively. The results indicate that although the binding specificity may be guided by molecular shape and H-bond interaction, the charge and hydrophobic interactions contributed most to the binding affinity.

Synthesis of sugar-amino acid-nucleosides as potential glycosyltransferase inhibitors.

Sugar-amino acid-nucleosides (SAAN) were synthesized to mimic glycosyl nucleotide donors based on the hypothesis that a basic amino acid may interact with carboxylate groups of the enzyme in a manner similar to the diphosphate metal ion complex. C-Glycoside analogues of the d-galactopyranose or l-arabinofuranose ring systems, and four amino acids (lysine, glutamine, tryptophan, and histidine), were chosen for this study. The targets were synthesized and tested against GlfT2, a galactofuranosyltransferase essential for cell wall galactan biosynthesis in Mycobacterium tuberculosis. The inhibition assay showed that analogues containing histidine and tryptophan are moderate inhibitors of GlfT2.

One-pot synthesis of highly functionalized morphans from C-glycosides.

Highly functionalized morphan derivatives were synthesized from nitroalkene 2'-(oxoalkyl)-C-glycosides by a tandem reaction that created three (two C-N and one C-C) new bonds and four stereogenic centers in a one-pot procedure under very mild conditions without the use of expensive reagents. The transformation was achieved from a beta-elimination/Michael addition cascade, followed by Michael addition of the amine and intramolecular enamination. In the presence of sodium cyanoborohydride the iminium intermediate was reduced in situ to afford the desired morphans.

Synthesis and biological evaluation of ether bridged bicyclic iminosugar derivatives.

Bicyclic iminosugar derivatives with an ether bridge bearing different substituents on C-2 and the nitrogen atom have been synthesized from a C-glycoside bearing an isopropylidene acetal. The activities of these compounds were investigated against several glycosidase enzymes and showed moderate inhibition and activation.

Triazole-fused sugars from nitroalkene-containing C-glycosides by a tandem 1,3-dipolar cycloaddition and intramolecular Michael addition.

A synthetic method to triazole-fused sugars by treatment of nitroalkene-containing C-glycosides with sodium azide is described. Initial experiments conducted at room temperature gave only the 1,3-dipolar cycloaddition products. However, at elevated reaction temperature the tandem beta-elimination/cycloaddition/Michael addition yielded 1,5-disubstituted triazole-fused sugars.

Synthesis of iminoalditol analogues of galactofuranosides and their activities against glycosidases.

Iminoalditol analogues of galactofuranosides were synthesized from 1-C-(2'-oxo-propyl)-1,4-dideoxy-1,4-imino-d-galactosides and different amines by reductive amination, followed by removal of protecting groups. The activity of these compounds against galactosidases and other glycosidases was investigated. The best inhibitor against beta-galactosidase (bovine liver) is a diastereomeric mixture of an iminoalditol (10h), which contains a hydrophobic hexadecyl aglycon (R=C(16)H(33)), whereas no significant inhibitory activity was observed with compounds having a hydrophilic aglycon. Surprisingly, activation of alpha-galactosidase (coffee bean) by 10h was also observed. Because these results were obtained from a mixture of iminoalditols, the inhibition and activation of glycosidases could result from different diastereomers.

Polyhydroxylated indolines and oxindoles from C-glycosides via sequential Henry reaction, Michael addition, and reductive amination/amidation.

6-nitro-2'-carbonyl-C-glycofuranosides synthesized via Henry reaction from 1-C-allyl 5-aldo-C-glycoside underwent an intramolecular Michael addition to afford nitrocyclohexanol derivatives in good to excellent yield. Reduction of the nitro group followed by intramolecular amination with ketone and aldehyde and amidation with ester produced indoline and oxindole derivatives, respectively, in excellent yield.

Stereoselective synthesis of polyhydroxylated quinolizidines from C-glycosides by one-pot double-conjugate addition.

An effective one-pot synthesis of polyhydroxylated quinolizidines from 1-C-(2'-oxo-4'-pentenyl)-5-azido-C-glycofuranosides was developed. Reduction of the 5-azido group using triphenylphosphine followed by base treatment produced quinolizidines in good yield. The base-mediated ring-opening beta-elimination produced an acyclic alpha,beta-conjugated ketone as a Michael acceptor, which was followed by an intramolecular nitrogen conjugate addition to form an aza-C-glycopyranoside intermediate. Meanwhile, the beta,gamma-double bond of the aglycon migrated under the basic conditions to form another alpha,beta-conjugated ketone. The subsequent intramolecular conjugate addition by the azasugar nitrogen led to the formation of the quinolizidines in a highly stereoselective manner. The stereoselectivity of the first conjugate addition giving azasugar is affected by the stereochemistry of the monosaccharide substrate, whereas the stereoselectivity in the second conjugate addition was likely directed entirely by steric repulsion from the azasugar.

Steady-state kinetics and molecular evolution of Escherichia coli MenD [(1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase], an anomalous thiamin diphosphate-dependent decarboxylase-carboligase.

(1R,6R)-2-Succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) synthase, or MenD, catalyzes the thiamin diphosphate- (ThDP-) dependent decarboxylation of 2-oxoglutarate, the subsequent addition of the resulting succinyl-ThDP moiety to isochorismate, and the delta-elimination of pyruvate to yield SHCHC, pyruvate, and carbon dioxide. The enzyme is part of a superfamily of ThDP-dependent 2-oxo acid decarboxylases that includes pyruvate decarboxylase, benzoylformate decarboxylase, and acetohydroxy acid synthase, among others. However, this is the only enzyme known to catalyze a Stetter-like 1,4-addition of a ThDP adduct to the beta-carbon of an unsaturated carboxylate. Herein we report properties of the MenD protein from Escherichia coli, including the results of the first steady-state kinetic studies of the SHCHC synthase reaction. The protein is a dimer and shows cooperativity with respect to both substrates. The enzyme prefers divalent manganese as its metal ion cofactor and shows no dependence on FAD. MenD, required for biosynthesis of menaquinone and phylloquinone, is found in the genomes of a wide range of bacteria, as well as that of the archaeon Halobacterium sp. NRC-1 and the eukaryote Arabidopsis thaliana. Sequence alignments with other members of the superfamily are used to predict amino acid residues likely to be important in the binding and activation of ThDP. A site-directed mutant that replaces the conserved glutamic acid residue (E55), predicted to interact with N1' of the aminopyrimidine ring, with glutamine was generated, with catastrophic results for catalysis. There is no evidence for the release of succinate semialdehyde as a product; therefore, EC 4.1.1.71 should not be used for this enzyme.