Selective Monovalent Galectin-8 Ligands Based on 3-Lactoylgalactoside.

Galectin-8 has gained attention as a potential new pharmacological target for the treatment of various diseases, including cancer, inflammation, and disorders associated with bone mass reduction. To that end, new molecular probes are needed in order to better understand its role and its functions. Herein we aimed to improve the affinity and target selectivity of a recently published galectin-8 ligand, 3-O-[1-carboxyethyl]-ß-d-galactopyranoside, by introducing modifications at positions 1 and 3 of the galactose. Affinity data measured by fluorescence polarization show that the most potent compound reached a KD of 12 µM. Furthermore, reasonable selectivity versus other galectins was achieved, making the highlighted compound a promising lead for the development of new selective and potent ligands for galectin-8 as molecular probes to examine the protein's role in cell-based and in vivo studies.

Protected N-Acetyl Muramic Acid Probes Improve Bacterial Peptidoglycan Incorporation via Metabolic Labeling.

Metabolic glycan probes have emerged as an excellent tool to investigate vital questions in biology. Recently, methodology to incorporate metabolic bacterial glycan probes into the cell wall of a variety of bacterial species has been developed. In order to improve this method, a scalable synthesis of the peptidoglycan precursors is developed here, allowing for access to essential peptidoglycan immunological fragments and cell wall building blocks. The question was asked if masking polar groups of the glycan probe would increase overall incorporation, a common strategy exploited in mammalian glycobiology. Here, we show, through cellular assays, that E. coli do not utilize peracetylated peptidoglycan substrates but do employ methyl esters. The 10-fold improvement of probe utilization indicates that (i) masking the carboxylic acid is favorable for transport and (ii) bacterial esterases are capable of removing the methyl ester for use in peptidoglycan biosynthesis. This investigation advances bacterial cell wall biology, offering a prescription on how to best deliver and utilize bacterial metabolic glycan probes.

Enzymatic synthesis and semi-preparative isolation of N-acetylmuramic acid 6-phosphate.

N-acetylmuramic acid 6-phosphate (MurNAc-6P) is a constituent of the bacterial peptidoglycan cell wall, serving as an anchor point of secondary cell wall polymers such as teichoic acids, and it is a key metabolite of the peptidoglycan recycling metabolism. Thus, there is a demand for MurNAc-6P as a standard for cell wall compositional and metabolic analyses and, in addition, as a substrate for peptidoglycan recycling enzymes, e.g. MurNAc-6P etherases (MurQ) and MurNAc-6P phosphatases (MupP), or as an effector molecule of transcriptional MurR regulators. However, MurNAc-6P is commercially not available. We report here the facile enzymatic production of MurNAc-6P in mg-scale from MurNAc and ATP, applying Clostridium acetobutylicum kinase MurK, and purification by semi-preparative HPLC. MurNAc-6P was quantified using a coupled enzyme assay, revealing 75-80% overall product yield, and high purity was confirmed by mass spectrometry and proton NMR.

1,6-AnhMurNAc derivatives for assay development of amidase AmiD.

Various peptidoglycan fragments were synthesized from two anhydro-muramic acid derivatives protected with a Bn or a PMB group at the 4th position, in homogenate phase or on a solid support. In order to facilitate HPLC detection, a chromophoric group was attached to the peptide chain. The periplasmic amidase sAmiD of Escherichia coli was used to cleave the amide bond between the lactyl group of the MurNAc and the α-amino group of L-Ala where the peptide chain was at least a dipeptide (L-Ala-γ-D-Glu) amidated by benzylamine on the γ-carboxyl group of D-Glu. In the presence of a tripeptide chain (L-Ala-γ-D-Glu-L-Lys) or a tetrapeptide chain (L-Ala-γ-D-Glu-m-A(2)pm-D-Ala) higher hydrolysis rates were observed. We have also demonstrated that the presence of TNB on the ε-amino group of L-Lys only has a small influence on the hydrolysis capacity of sAmiD.

The first direct oxidative conversion of a selenol to a stable selenenic acid: experimental demonstration of three processes included in the catalytic cycle of glutathione peroxidase.

[structure: see text]. A stable selenenic acid was synthesized by direct oxidation of a selenol bearing a novel bowl-type substituent with H2O2, and its structure was established by X-ray crystallographic analysis. Selenenyl sulfides obtained by the reaction of the selenenic acid with 1,4-dithiols were reduced to the corresponding selenol by treatment with a tertiary amine, thus achieving the experimental demonstration of three processes included in the catalytic cycle of glutathione peroxidase.

Synthesis and biological evaluation of analogues of bacterial lipid I.

Bacterial Lipid I analogues containing different anomeric groups at the muramic acid moiety were synthesized and screened in MurG enzyme assays run in the presence and absence of cell wall membranes. The results obtained in this study help elucidate the role of the lipid diphosphate in the recognition of Lipid I by MurG.

Muramic acid derivatives as glycosyl donors for the synthesis of muramyl-containing glycosphingolipids and fatty acids.

2-Azido-2-deoxy-4,6-O-isopropylidene-3-O-[(1R)-(methoxycarbonyl)ethyl]- alpha-D-glucopyranosyl trichloroacetimidate (3 alpha) has been used as the glycosyl donor in the synthesis of glycosphingolipids 14 and 27. Reaction of 3 alpha with (2S, 3R, 4E)-2-azido-3-benzoyloxy-4-octadecen-1-ol (6) gave (2S, 3R, 4E)-2-azido-1-(2-azido-2-deoxy-4,6-O-isopropylidene-3-O-[(1R)-1-(m ethoxycarbonyl)ethyl]-beta-D-glucopyranosyloxyl)-3-benzoyloxy-4- octadecene (7), which was converted into (2S, 3R, 4E)-1-(2-deoxy-2-hexadecanoylamino-3-O-[(2R)-propanoyl-(L-alanyl-D -isoglutamine benzyl ester)-2-yl]-beta-D-glucopyranosyloxy)-2-hexadecanoylamino-4-oc tadecen-ol (14). Reaction of 3 alpha with tert-butyldimethylsilyl 2-azido-3,6-di-O-benzyl-2-deoxy-beta-D-glucopyranoside (15) gave tert-butyldimethylsilyl 2-azido-4-O-(2-azido-2-deoxy-4,6-O-isopropylidene-3-O-[(1R)-1-(methox ycarbonyl)ethyl]-beta-D-glucopyranosyl)-3,6-di-O-benzyl-2-deoxy-be ta- D-gluc opyranoside (16 beta), which was converted into 1,3,6-tri-O-acetyl-2-deoxy-4-O-(4,6-di-O-acetyl-2-deoxy-2-hexadecanoy lam ino-3-O-[2R)-propanoyl-(L-alanyl-D-isoglutamine methyl ester)-2-yl]-beta-D-glucopyranosyl)-2-hexadecanoylamino-D-glucopyranose (27).

The macrophage, target cell of the synthetic adjuvant muramyl dipeptide.

The mechanism of adjuvant activity of the synthetic glycopeptide N-acetylmuramul-L-alanyl-D-isoglutamine or muramyl dipeptide (MDP) was studied using in vitro plaque-forming cell (PFC) response to sheep erythrocytes (SRBC). Addition of MDP to DBA/2 mouse spleen cell cultures resulted regularly in a 2 to 3-fold increase of PFC numbers/10(6) recovered cells (p less than 0.01). Supernates (SPN) from MDP-stimulated cultures added to standard spleen cell + SRBC cultures brought about even more important increases of PFC numbers (p less than 0.01 to p less than 0.001). SPN from cultures supplemented with MDP alone (without SRBC) were more active than those of cell + MDP + SRBC cultures, and SPN removed on day 3 of culture were more active than those of day 5. This activity of SPN was maintained accross an H-2 histocompatibility barrier. Although pretreatment of spleen cells with anti-theta antigen serum entirely suppressed the anti-SRBC PFC response in spite of the presence of MDP, SPN from these cultures were as active as SPN from normal spleen cell MDP-stimulated cultures. In contrast, pretreatment of spleen cells with specific rabbit anti-mouse macrophage serum entirely suppressed both anti-SRBC response and SPN activity. It was concluded that the target cell for MDP is the macrophage which releases factors ultimately acting on B cells through T cell mediation.